Methods and compositions for enhancing immune responses

ABSTRACT

Compositions and methods are described for generating an improved effective immune response against an immunogen in humans. The enhanced immune response, is obtained by using an MVA vector as a prime and an adenovirus vector as a boost and is characterized by a high level of antibody response specific to the immunogen, and an enhanced cellular immune response. The compositions and methods can be used to provide a protective immunity against a disease, such as an infection of one or more subtypes of Ebola and Marburg filoviruses, in humans.

FIELD OF THE INVENTION

This invention relates to methods and compositions for enhancing animmune response in a human subject. In particular, the methods andcompositions provide a strong induction of B cell and T cell activityagainst an immunogen in a human subject, which can be used to provide aneffective treatment and/or protection against a disease, such as a tumoror an infectious disease, more particularly an infection by a filovirus,in the human subject.

BACKGROUND OF THE INVENTION

Vaccines can be used to provide immune protection against pathogens,such as viruses, bacteria, fungi, or protozoans, as well as cancers.

Infectious diseases are the second leading cause of death worldwideafter cardiovascular disease but are the leading cause of death ininfants and children (Lee and Nguyen, 2015, Immune Network, 15(2):51-7).Vaccination is the most efficient tool for preventing a variety ofinfectious diseases. The goal of vaccination is to generate apathogen-specific immune response providing long-lasting protectionagainst infection. Despite the significant success of vaccines,development of safe and strong vaccines is still required due to theemergence of new pathogens, re-emergence of old pathogens and suboptimalprotection conferred by existing vaccines. Recent important emerging orre-emerging diseases include: severe acute respiratory syndrome (SARS)in 2003, the H1N1 influenza pandemic in 2009, and Ebola virus in 2014.As a result, there is a need for the development of new and effectivevaccines against emerging diseases.

Cancer is one of the major killers in the Western world, with lung,breast, prostate, and colorectal cancers being the most common(Butterfield, 2015, BMJ, 350:h988). Several clinical approaches tocancer treatment are available, including surgery, chemotherapy,radiotherapy, and treatment with small molecule signaling pathwayinhibitors. Each of these standard approaches has been shown to modulateantitumor immunity by increasing the expression of tumor antigens withinthe tumor or causing the release of antigens from dying tumor cells andby promoting anti-tumor immunity for therapeutic benefit. Immunotherapyis a promising field that offers alternative methods for treatment ofcancer. Cancer vaccines are designed to promote tumor-specific immuneresponses, particularly cytotoxic CD8+ T cells that are specific totumor antigens. Clinical efficacy must be improved in order for cancervaccines to become a valid alternative or complement to traditionalcancer treatments. Considerable efforts have been undertaken so far tobetter understand the fundamental requirements for clinically-effectivecancer vaccines. Recent data emphasize that important requirements,among others, are (1) the use of multi-epitope immunogens, possiblyderiving from different tumor antigens; (2) the selection of effectiveadjuvants; (3) the association of cancer vaccines with agents able tocounteract the regulatory milieu present in the tumor microenvironment;and (4) the need to choose the definitive formulation and regimen of avaccine after accurate preliminary tests comparing different antigenformulations (Fenoglio et al., 2013, Hum Vaccin Immunother,(12):2543-7). A new generation of cancer vaccines, provided with bothimmunological and clinical efficacy, is needed to address theserequirements.

Ebolaviruses, such as Zaire ebolavirus (EBOV) and Sudan ebolavirus(SUDV), and the closely related Marburg virus (MARV), are associatedwith outbreaks of highly lethal Ebola Hemorrhagic Fever (EHF) in humansand primates in North America, Europe, and Africa. These viruses arefiloviruses that are known to infect humans and non-human primates withsevere health consequences, including death. Filovirus infections haveresulted in case fatality rates of up to 90% in humans. EBOV, SUDV, andMARV infections cause EHF with death often occurring within 7 to 10 dayspost-infection. EHF presents as an acute febrile syndrome manifested byan abrupt fever, nausea, vomiting, diarrhea, maculopapular rash,malaise, prostration, generalized signs of increased vascularpermeability, coagulation abnormalities, and dysregulation of the innateimmune response. Much of the disease appears to be caused bydysregulation of innate immune responses to the infection and byreplication of virus in vascular endothelial cells, which induces deathof host cells and destruction of the endothelial barrier. Filovirusescan be spread by small particle aerosol or by direct contact withinfected blood, organs, and body fluids of human or NHP origin.Infection with a single virion is reported to be sufficient to causeEbola hemorrhagic fever (EHF) in humans. Presently, there is notherapeutic or vaccine approved for treatment or prevention of EHF.Supportive care remains the only approved medical intervention forindividuals who become infected with filoviruses.

As the cause of severe human disease, filoviruses continue to be ofconcern as both a source of natural infections, and also as possibleagents of bioterrorism. The reservoir for filoviruses in the wild hasnot yet been definitively identified. Four subtypes of Ebolaviruses havebeen described to cause EHF, i.e., those in the Zaire, Sudan, Bundibugyoand Ivory Coast episodes (Sanchez A. et al., 1996, PNAS USA,93:3602-3607). These subtypes of Ebolaviruses have similar geneticorganizations, e.g., negative-stranded RNA viruses containing sevenlinearly arrayed genes. The structural gene products include, forexample, the envelope glycoprotein that exists in two alternative forms,a secreted soluble glycoprotein (ssGP) and a transmembrane glycoprotein(GP) generated by RNA editing that mediates viral entry (Sanchez A. etal., 1996, PNAS USA, 93:3602-3607).

It has been suggested that immunization can be useful in protectingagainst Ebola infection because there appears to be less nucleotidepolymorphism within Ebola subtypes than among other RNA viruses (SanchezA. et al., 1996, PNAS USA, 93:3602-3607). Until recently, developmentsof preventive vaccines against filoviruses have had variable results,partly because the requirements for protective immune responses againstfilovirus infections are poorly understood. Additionally, the largenumber of filoviruses circulating within natural reservoirs complicatesefforts to design a vaccine that protects against all species offiloviruses.

Currently, there are several vaccine antigen delivery platforms thatdemonstrated various levels of protection in non-human primates (NHPs)exposed with high infectious doses of filoviruses. Vaccine candidatesare in development based on a variety of platform technologies includingreplication competent vectors (e.g. Vesicular Stomatitis Virus; Rabiesvirus; Parainfluenza Virus); replication incompetent vectors(Adenovirus, Modified Vaccinia Ankara Virus); protein subunits inclusiveof Virus Like Particles expressed in bacterial cells, insect cells,mammalian cells, plant cells; DNA vaccines; and/or live and killedattenuated filovirus (Friedrich et al., 2012, Viruses, 4(9):1619-50).The EBOV glycoprotein GP is an essential component of a vaccine thatprotects against exposures with the same species of EBOV. Furthermore,inclusion of the GP from EBOV and SUDV, the two most virulent species ofebolaviruses, can protect monkeys against EBOV and SUDV intramuscularexposures, as well as exposures with the distantly related Bundibugyo(BDBV), Taï Forest ebolavirus (TAFV; formerly known as Ivory Coast orCote d'Ivoire) species. Likewise, inclusion of the GP from MARV canprotect monkeys against intramuscular and aerosol MARV exposures. Thedevelopment of medical countermeasures for these viruses is a highpriority, in particular the development of a PAN-filovirus vaccine—thatis one vaccine that protects against all pathogenic filoviruses.

Replication-defective adenovirus vectors (rAd) are powerful inducers ofcellular immune responses and have therefore come to serve as usefulvectors for gene-based vaccines particularly for lentiviruses andfiloviruses, as well as other nonviral pathogens (Shiver et al., 2002,Nature, 415(6869): 331-5; Hill et al., 2010, Hum Vaccin 6(1): 78-83;Sullivan et al., 2000, Nature, 408(6812): 605-9; Sullivan et al., 2003,Nature, 424(6949): 681-4; Sullivan et al., 2006, PLoS Med, 3(6): e177;Radosevic et al., 2007, Infect Immun, 75(8):4105-15; Santra et al.,2009, Vaccine, 27(42): 5837-45).

Adenovirus-based vaccines have several advantages as human vaccinessince they can be produced to high titers under GMP conditions and haveproven to be safe and immunogenic in humans (Asmuth et al., 2010, JInfect Dis 201(1): 132-41; Kibuuka et al., 2010, J Infect Dis 201(4):600-7; Koup et al., 2010, PLoS One 5(2): e9015; Catanzaro et al., 2006,J Infect Dis, 194(12): 1638-49; Harro et al., 2009, Clin VaccineImmunol, 16(9): 1285-92). While most of the initial vaccine work wasconducted using rAd5 due to its significant potency in eliciting broadantibody and CD8+ T cell responses, pre-existing immunity to rAd5 inhumans may limit efficacy (Catanzaro et al., 2006, J Infect Dis,194(12): 1638-49; Cheng et al., 2007, PLoS Pathog, 3(2): e25; McCoy etal., 2007, J Virol, 81(12): 6594-604; Buchbinder et al., 2008, Lancet,372(9653): 1881-93). This property might restrict the use of rAd5 inclinical applications for many vaccines that are currently indevelopment including Ebolavirus (EBOV) and Marburg virus (MARV).

Replication-defective adenovirus vectors, rAd26 and rAd35, derived fromadenovirus serotype 26 and serotype 35, respectively, have the abilityto circumvent Ad5 pre-existing immunity. rAd26 can be grown to hightiters in Ad5 E1-complementing cell lines suitable for manufacturingthese vectors at a large scale and at clinical grade (Abbink, et al.,2007, J Virol, 81(9):4654-63), and this vector has been shown to inducehumoral and cell-mediated immune responses in prime-boost vaccinestrategies (Abbink, et al., 2007, J Virol, 81(9):4654-63; Liu et al.,2009, Nature, 457(7225): 87-91). rAd35 vectors grow to high titers oncell lines suitable for production of clinical-grade vaccines (Havengaet al., 2006, J Gen Virol, 87: 2135-43), and have been formulated forinjection as well as stable inhalable powder (Jin et al., 2010, Vaccine28(27): 4369-75). These vectors show efficient transduction of humandendritic cells (de Gruijl et al., 2006, J Immunol, 177(4): 2208-15;Lore et al., 2007, J Immunol, 179(3): 1721-9), and thus have thecapability to mediate high level antigen delivery and presentation.

Modified Vaccinia Ankara (MVA) virus is related to Vaccinia virus, amember of the genera Orthopoxvirus in the family of Poxviridae.Poxviruses are known to be good inducers of CD8 T cell responses becauseof their intracytoplasmic expression. However, they may be poor atgenerating CD4 MHC class II restricted T cells (see for example Haslettet al., 2000, Journal of Infectious Diseases, 181: 1264-72, page 1268).MVA has been engineered for use as a viral vector for recombinant geneexpression or as recombinant vaccine.

Strains of MVA having enhanced safety profiles for the development ofsafer products, such as vaccines or pharmaceuticals, have been developedby Bavarian Nordic. MVA was further passaged by Bavarian Nordic and isdesignated MVA-BN, a representative sample of which was deposited onAug. 30, 2000 at the European Collection of Cell Cultures (ECACC) underAccession No. V00083008. MVA-BN is further described in WO 02/42480 (US2003/0206926) and WO 03/048184 (US 2006/0159699), both of which areincorporated by reference herein in their entirety.

MVA-BN can attach to and enter human cells where virally-encoded genesare expressed very efficiently. MVA-BN is replication incompetent,meaning that the virus does not replicate in human cells. In humancells, viral genes are expressed, and no infectious virus is produced.MVA-BN is classified as Biosafety Level 1 organism according to theCenters for Disease Control and Prevention in the United States.Preparations of MVA-BN and derivatives have been administered to manytypes of animals, and to more than 2000 human subjects, includingimmune-deficient individuals. All vaccinations have proven to begenerally safe and well tolerated. Despite its high attenuation andreduced virulence, in preclinical studies MVA-BN has been shown toelicit both humoral and cellular immune responses to vaccinia and toheterologous gene products encoded by genes cloned into the MVA genome[E. Harrer et al. (2005), Antivir. Ther. 10(2):285-300; A. Cosma et al.(2003), Vaccine 22(1):21-9; M. Di Nicola et al. (2003), Hum. Gene Ther.14(14):1347-1360; M. Di Nicola et al. (2004), Clin. Cancer Res.,10(16):5381-5390].

Protective immunity to infection relies on both the innate and adaptiveimmune response. The adaptive immune response includes production ofantibodies by B cells (humoral immune response) and the cytotoxicactivity of CD8+ effector T cells (cellular immune response) and CD4+ Tcells, also known as helper T cells, who play a key role in both theImmoral and the cellular immune response.

CD4+ T cells are stimulated by antigens to provide signals that promoteimmune responses. CD4+ T cells act through both cell-cell interactionsand the release of cytokines to help trigger B cell activation andantibody production, activation and expansion of cytotoxic CD8+ T cells,and macrophage activity.

Antibody-mediated protection can be extraordinarily long-lived, andneutralizing antibodies present at the time of pathogen encounter canprevent rather than combat infection, thereby achieving ‘sterilizing’immunity (Swain et al., 2012, Nat Rev Immunol, 12(2): 136-148).Following viral infection, CD4+ signaling is necessary to direct theformation of germinal centers, where CD4+ cells promote B cell isotypeswitching and affinity maturation of antibody responses as well as thegeneration of B cell memory and long-lived antibody-producing plasmacells. Thus, CD4+ cells are likely to be important for generatinglong-lived antibody responses and protective immunity to most, if notall, pathogens.

The role of CD4+ T cells in helping the priming, effector function, andmemory of CD8+ T cells is especially important in the case of chronicinfections, when CD8+ T cells rely on continued rounds of expansion, forwhich CD4+ T cell cytokine production is critical (Swain et al., 2012,Nat Rev Immunol, 12(2): 136-148).

Recent data has indicates that the role of CD4+ T cells extend furtherthan that of cytokine production. For example, CD4+ T cells can recruitkey lymphoid populations into secondary lymphoid tissue or sites ofpathogen infection (Sant and McMichael, 2012, J Exp Med, 209(8):1391-5).Specifically, CD4+ T cells can promote engagement of CD8+T cells withdendritic cells in secondary lymphoid tissue, cause influx of lymphoidcells into draining lymph nodes, and recruit effectors to the site ofviral replication. In addition, CD4+ T cells can also protect againstpathogens through direct cytolytic activity.

Following the resolution of primary immune responses, or aftersuccessful vaccination, most pathogen-specific effector CD4+ T cellsdie, leaving behind a small population of long-lived memory cells.Memory CD4+ T cells enhance early innate immune responses followinginfections in the tissues that contribute to pathogen control (Swain etal., 2012, Nat Rev Immunol, 12(2): 136-148). Importantly, CD4+ T cellsprovide more rapid help to B cells, and potentially to CD8+ T cells,thereby contributing to a faster and more robust immune response.

The range of functions of CD4+ T cells during an immune responsehighlights their key role in generating highly effective immuneprotection against pathogens. Recent studies have provided new evidencefor CD4+ T cells as direct effectors in antiviral immunity (Sant et al.,2012, J. Exp. Med. 209: 1391-1395). Preexisting influenza-specific CD4+T cells were reported to correlate with disease protection againstinfluenza challenge in humans (Wilkinson et al., 2012, Nature Medicine,18: 274-280).

Several assays are used to detect immune responses, including, e.g.,ELISA (enzyme-linked immunosorbent assay), ELISPOT (enzyme-linkedimmunospot), and ICS (intracellular cytokine staining). ELISA assaysanalyze, e.g., levels of secreted antibodies or cytokines. When ELISAassays are used to determine levels of antibodies that bind to aparticular antigen, an indicator of the humoral immune response, theymay also reflect CD4+ T cell activity, as the production ofhigh-affinity antibodies by B cells depends on the activity of CD4+helper T cells. ELISPOT and ICS are single-cell assays that analyze,e.g., T cell responses to a particular antigen. ELISPOT assays measurethe secretory activity of individual cells, and ICS assays analyzelevels of intracellular cytokine. CD4+ specific and CD8+ specific T cellresponses can be determined using ICS assays.

There are published papers testing methods for using MVA-Ad prime-boostregimens in animals, such as monkeys and mice. However, no MVA-Adprime-boost regimen has been shown to be more effective at stimulatingan immune response than the complementary Ad-MVA prime-boost regimenuntil now. For example, Barouch et al. (2012, Nature, 482(7383):89-93)found that, in monkeys, a heterologous regimen comprising MVA/M26 was“comparatively less efficacious than Ad26/MVA or Ad35/Ad26, whichreduced viral load set-points by greater than 100-fold.” In particular,the cellular immune response to SIV Gag, Pol, and Env in rhesus monkeyswas less-pronounced for the MVA/Ad26 prime-boost regimen administered ona 0-24 week schedule than for the opposite Ad26/MVA regimen, as measuredby IFN-gamma ELISPOT and ICS assays. The antibody response was also lesseffective for the MVA/Ad26 regimen than for the Ad26/MVA regimen, asevidenced by an ELISA assay, though to a lesser extent. Roshorm et al.(2012, Eur J Immunol, 42(12):3243-55) found that an MVA/ChAdV68prime-boost regimen administered in mice on a 0-4 week schedule was nomore effective at inducing an immune response to HIV Gag than theopposite ChAdV68/MVA regimen, as measured by an ICS assay for CD8+ Tcell activity. Gilbert et al. (2002, Vaccine, 20(7-8):1039-45) foundthat an MVA/Ad5 prime-boost regimen administered in mice on a 0-14 dayschedule was slightly less effective in producing an immune response toPlasmodium CS than the opposite Ad5/MVA regimen, as measured by anELISPOT assay. The MVA/Ad5 regimen was even less effective than theAd5/MVA regimen when both were administered on a 0-10 day schedule.Additionally, the MVA/Ad5 regimen was less effective in protectingimmunized mice against a challenge infection (80% vs. 100% protection).None of these reports indicate that an MVA/Ad regimen can result in astronger humoral and/or cellular immune response in humans, than anAd/MVA regimen.

There is an unmet need for improved vaccines that elicit broad andstrong immune responses in humans against antigenic proteins, andparticularly vaccines that provide protective immunity against thedeadly Ebola and Marburg filoviruses.

BRIEF SUMMARY OF THE INVENTION

It is now discovered, for the first time, that a specific order ofadministration of prime-boost regimens of replication incompetentvectors generates an improved effective immune response that could beapplied to provide treatment and/or protection against a disease, suchas a tumor or an infectious disease, more particularly an infection by afilovirus, in a human subject. Surprisingly, it has now been found thatdifferent from the previously reported animal studies, use of an MVAvector as a prime and an adenovirus vector as a boost generates asuperior immune response against an immunogen, characterized by a stronginduction of T cell activity and a high level of antibody responsespecific to the immunogen.

In certain embodiments of the invention, MVA-prime and adenovirus-boostcombinations of replication incompetent vectors generate an enhancedimmune response to an antigenic protein or an immunogenic polypeptidethereof in a human subject. The antigenic protein or immunogenicpolypeptide thereof can be any antigenic protein or immunogenicpolypeptide thereof. For example, the antigenic protein or immunogenicpolypeptide thereof can be derived from a pathogen, e.g., a virus, abacterium, a fungus, a protozoan, or a tumor.

Accordingly, one general aspect of the invention relates to a method ofenhancing an immune response in a human subject, the method comprising:

-   -   a. administering to the human subject a first composition        comprising an immunologically effective amount of a MVA vector        comprising a first polynucleotide encoding an antigenic protein        or an immunogenic polypeptide thereof for priming the immune        response; and    -   b. administering to the subject a second composition comprising        an immunologically effective amount of an adenovirus vector        comprising a second polynucleotide encoding the antigenic        protein or an immunogenic polypeptide thereof for boosting the        immune response;    -   to thereby obtain an enhanced immune response against the        antigenic protein in the human subject.

In a preferred embodiment of the invention, the enhanced immune responsecomprises an enhanced antibody response against the antigenic protein inthe human subject.

In a preferred embodiment of the invention, the enhanced immune responsecomprises an enhanced CD4+ and/or CD8+ T cell response against theantigenic protein in the human subject.

Another aspect of the invention relates to a method of eliciting animmune response in a human subject, the method comprising:

-   -   a. administering to the human subject a first composition        comprising an immunologically effective amount of a MVA vector        comprising a first polynucleotide encoding an antigenic protein        or an immunogenic polypeptide thereof for priming the immune        response; and    -   b. administering to the subject a second composition comprising        an immunologically effective amount of an adenovirus vector        comprising a second polynucleotide encoding the antigenic        protein or an immunogenic polypeptide thereof for boosting the        immune response;    -   to thereby obtain an enhanced immune response in the human        subject relative to the immune response that would be observed        if the second composition would be administered for priming and        the first composition would be administered for boosting the        immune response.

In a preferred embodiment of the invention, the enhanced immune responsegenerated by the method comprises an enhanced antibody response againstthe antigenic protein in the human subject. Such a response can e.g. becharacterized by the presence of a high proportion of responders, suchas more than 50%, 60%, 70%, 80%, 90%, or 100% of subjects tested.

In one embodiment of the invention, the enhanced immune responsegenerated by the method comprises an enhanced CD8+ T cell responseagainst the antigenic protein in the human subject [e.g. a responsecharacterized by the presence of a high proportion of CD8+ responders,such as more than 50%, 60%, 70%, 80%, 90%, or 100% of subjects tested asdetermined by an ICS assay, with a median total cytokine response ofabout 0.1%, 0.2%, 0.3%, 0.4%, 0.5% or more]. In another embodiment ofthe invention, the enhanced CD8+ T cell response generated by the methodcomprises an increase or induction of poly functional CD8+ T cellsspecific to the antigenic protein. Such polyfunctional CD8+ T cellsexpress more than one cytokine, such as two or more of IFN-gamma, IL-2and TNF-alpha.

In one embodiment of the invention, the enhanced immune responsegenerated by the method comprises an enhanced CD4+ T cell responseagainst the antigenic protein in the human subject [e.g. a responsecharacterized by the presence of a high proportion of CD4+ responders,such as more than 50%, 60%, 70%, 80%, 90%, or 100% of subjects tested asdetermined by an ICS assay, with a median total cytokine response ofabout 0.1%, 0.2%, 0.3%, 0.4%, 0.5% or more]. In another embodiment ofthe invention, the enhanced CD4+ T cell response generated by the methodcomprises an increase or induction of polyfunctional CD4+ T cellsspecific to the antigenic protein. Such polyfunctional CD4+ T cellsexpress more than one cytokine, such as two or more of IFN-gamma, IL-2and TN F-alpha.

In another preferred embodiment of the invention, the enhanced immuneresponse further comprises an enhanced antibody response against theantigenic protein in the human subject. Such a response can e.g. becharacterized by the presence of a high proportion of responders, suchas more than 50%, 60%, 70%, 80%, 90%, or 100% of subjects tested. Inanother embodiment of the invention, the enhanced immune responsefurther comprises an enhanced CD8+ T cell response against the antigenicprotein in the human subject [e.g. a response characterized by thepresence of a high proportion of CD8+responders, such as more than 50%,60%, 70%, 80%, 90%, or 100% of subjects tested as determined by an ICSassay, with a median total cytokine response of about 0.1%, 0.2%, 0.3%,0.4%, 0.5% or more]. In one embodiment of the invention, the enhancedCD8+ T cell response generated by the method comprises an increase orinduction of polyfunctional CD8+ T cells specific to the antigenicprotein in the human subject.

In a more preferred embodiment of the invention, the enhanced immuneresponse comprises an enhanced CD4+ T cell response, an enhancedantibody response and an enhanced CD8+ T cell response, against theantigenic protein in the human subject.

In a preferred embodiment of the invention, the adenovirus vector is arAd26 vector.

In another preferred embodiment of the invention, the boosting step (b)is conducted 1-12 weeks after the priming step (a). In yet anotherembodiment of the invention, the boosting step (b) is repeated one ormore times after the initial boosting step.

In a preferred embodiment of the invention, the boosting step (b) isconducted 2-12 weeks after the priming step (a). In another preferredembodiment of the invention, the boosting step (b) is conducted 4-12weeks after the priming step (a). In another preferred embodiment of theinvention, the boosting step (b) is conducted 1 week after the primingstep (a). In another preferred embodiment of the invention, the boostingstep (b) is conducted 2 weeks after the priming step (a). In anotherpreferred embodiment of the invention, the boosting step (b) isconducted 4 weeks after the priming step (a). In another preferredembodiment of the invention, the boosting step (b) is conducted 8 weeksafter the priming step (a).

In an embodiment of the invention, the antigenic protein is derived froma pathogen, such as a virus, a bacterium, a fungus, or a protozoan. Inanother embodiment of the invention, the antigenic protein is derivedfrom a tumor, preferably a cancer.

In an embodiment of the invention, the first polynucleotide and thesecond polynucleotide encode for the same antigenic protein orimmunogenic polypeptide thereof. In another embodiment of the invention,the first polynucleotide and the second polynucleotide encode fordifferent immunogenic polypeptides or epitopes of the same antigenicprotein. In yet another embodiment of the invention, the firstpolynucleotide and the second polynucleotide encode for different, butrelated, antigenic proteins or immunogenic polypeptide thereof. Forexample, the related antigenic proteins can be substantially similarproteins derived from the same antigenic protein, or different antigenicproteins derived from the same pathogen or tumor.

According to embodiment of the invention, a method of the inventionprovides a protective immunity to the human subject against a diseaseassociated with the antigenic protein, such as a tumor or an infectiousdisease.

In one preferred embodiment, the prime-boost combination of replicationincompetent MVA and adenovirus vectors enhances a protective immuneresponse against a tumor in a human subject.

In another preferred embodiment, the prime-boost combination ofreplication incompetent MVA and adenovirus vectors enhances an immuneresponse against a pathogen, more preferably one or more filovirussubtypes, such as the Ebola and/or Marburg filoviruses, in a humansubject.

The filovirus subtypes according to the invention can be any filovirussubtype. In a preferred embodiment, the filovirus subtypes are selectedfrom the group of Zaire, Sudan, Reston, Bundibugyo, Taï Forest andMarburg. The antigenic proteins can be any protein from any filoviruscomprising an antigenic determinant. In a preferred embodiment theantigenic proteins are glycoproteins or nucleoproteins. The antigenicproteins encoded by the MVA vectors or adenovirus vectors comprised inthe first and second composition according to the invention can be anyantigenic protein from any filovirus.

In another preferred embodiment, the MVA vector in the first compositioncomprises a nucleic acid encoding antigenic proteins of at least fourfilovirus subtypes. Preferably, the MVA vector comprises a nucleic acidencoding one or more antigenic proteins having the amino acid sequencesof SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5. Mostpreferably, the MVA vector comprises a nucleic acid encoding fourantigenic proteins having the amino acid sequences of SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5.

In certain embodiments, the second composition comprises at least oneadenovirus vector comprising a nucleic acid encoding an antigenicprotein of at least one filovirus subtype. The at least one filovirussubtype encoded by the adenovirus can be selected from any of the fourfilovirus subtypes encoded by the MVA vector, or a new subtype notencoded by the MVA vector. In a preferred embodiment, the antigenicprotein of the at least one filovirus subtype encoded by the adenovirusvector has the amino acid sequence selected from the group consisting ofSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.

In another embodiment, the second composition comprises more than oneadenovirus vectors, each comprising a nucleic acid encoding an antigenicprotein of at least one filovirus subtype. The antigenic proteinsencoded by the more than one adenovirus vectors can be the same ordifferent antigenic proteins. For example, the second composition cancomprise a first adenovirus vector comprising a nucleic acid encoding afirst antigenic protein having an amino acid sequence selected from thegroup consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4,and SEQ ID NO:5. The second composition can further comprise a secondadenovirus vector comprising a nucleic acid encoding a second antigenicprotein having an amino acid sequence selected from the group consistingof SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.The second composition can additionally comprise a third adenovirusvector comprising a nucleic acid encoding a third antigenic proteinhaving an amino acid sequence selected from the group consisting of SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5. Thefirst, second and third adenovirus vectors can be same or different. Thefirst, second and third antigenic proteins can be same or different.

In a preferred embodiment, the second composition comprises a firstadenovirus vector comprising a nucleic acid encoding an antigenicprotein having the amino acid sequence of SEQ ID NO:1. In anotherembodiment, the second composition further comprises a second adenovirusvector comprising a nucleic acid encoding an antigenic protein havingthe amino acid sequence of SEQ ID NO:2. In yet another embodiment, thesecond composition additional comprises a third adenovirus vectorcomprising a nucleic acid encoding an antigenic protein having the aminoacid sequence of SEQ ID NO:3.

It is contemplated that the methods, vaccines, and compositionsdescribed herein can be embodied in a kit. For example, in oneembodiment, the invention can include a kit comprising:

-   -   (a) a first composition comprising an immunologically effective        amount of a MVA vector comprising a nucleic acid encoding an        antigenic protein of a first filovirus subtype or a        substantially similar antigenic protein, together with a        pharmaceutically acceptable carrier; and    -   (b) a second composition comprising an immunologically effective        amount of an adenovirus vector comprising a nucleic acid        encoding antigenic proteins of at least one filovirus subtype or        a substantially similar antigenic protein, together with a        pharmaceutically acceptable carrier;    -   wherein composition (a) is a priming composition and        composition (b) is a boosting composition.

In a preferred embodiment, the invention relates to a combinationvaccine, a kit or a use wherein the MVA vector in the first compositioncomprises a nucleic acid encoding one or more antigenic proteins fromfour different filovirus subtypes having the amino acid sequences of SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5, preferably allfour of the antigenic proteins; and wherein the adenovirus vector in thesecond composition comprises a nucleic acid encoding an antigenicprotein having the amino acid sequence of SEQ ID NO: 1.

In yet another preferred embodiment, the invention relates to acombination vaccine, a kit or a use wherein the MVA vector incomposition (a) comprises a nucleic acid encoding one or more antigenicproteins from four different filovirus subtypes having SEQ ID NO: 1, SEQID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5, preferably all four of theantigenic proteins; and wherein the second composition comprises atleast one adenovirus comprising a nucleic acid encoding an antigenicprotein with SEQ ID NO: 1, at least one adenovirus comprising a nucleicacid encoding an antigenic protein with SEQ ID NO: 2, and at least oneadenovirus comprising a nucleic acid encoding an antigenic protein withSEQ ID NO: 3.

In a preferred embodiment, the adenovirus vectors comprised in thecombination vaccine or kit of the invention or the adenovirus vectorsused for generating a protective immune response against at least one ofthe filovirus subtypes, are rAd26 or rAd35 vectors.

In another preferred embodiment, the priming vaccination is conducted atweek 0, followed by a boosting vaccination at week 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, or later. Preferably, the boosting vaccination isadministered at week 1-10, more preferably at week 1, 2, 4 or 8.

According to embodiments of the invention, the boosting step (b) can berepeated one or more times after the initial boosting step. Theadditional boosting administration can be performed, for example, 6months, 1 year, 1.5 years, 2 years, 2.5 years, 3 years after the primingadministration, or later.

In a preferred embodiment of the invention, the method comprises apriming vaccination with an immunologically effective amount of one ormore MVA vectors expressing one or more filovirus glycoproteins,followed by a boosting vaccination with an immunologically effectiveamount of one or more adenovirus vectors, preferably Ad26 vectorsexpressing one or more filovirus glycoproteins or substantially similarglycoproteins.

In preferred embodiments of the invention, the one or more filovirusesare Ebolaviruses or Marburg viruses. The Ebolavirus can be of anyspecies, for example, Zaire ebolavirus (EBOV) and Sudan ebolavirus(SUDV), Reston, Bundibugyo, Taï Forest. The Marburg virus (MARV) can beof any species. Exemplary amino acid sequences of suitable filovirusantigenic proteins are shown in SEQ ID NO: 1 to SEQ ID NO: 5.

The invention also relates to use of the first and second compositionsaccording to embodiments of the invention for enhancing an immuneresponse in a human subject, wherein the first composition isadministered to the human subject for priming the immune response, andthe second composition is administered to the human subject for boostingthe immune response, to thereby obtain an enhanced immune responseagainst the antigenic protein in the human subject.

The invention further relates to:

-   -   a. a first composition comprising an immunologically effective        amount of a MVA vector comprising a first polynucleotide        encoding an antigenic protein or an immunogenic polypeptide        thereof; and    -   b. a second composition comprising an immunologically effective        amount of an adenovirus vector comprising a second        polynucleotide encoding the antigenic protein or an immunogenic        polypeptide thereof for boosting the immune response;    -   the first and second compositions for use in inducing an        enhanced immune response against the antigenic protein in a        human subject, wherein the first composition is administered to        the human subject for priming the immune response, and the        second composition is administered to the human subject one or        more times for boosting the immune response.

In one preferred embodiment, the antigenic protein or an immunogenicpolypeptide thereof encoded by the first polynucleotide is derived froma pathogen or a tumor. In another preferred embodiment, the antigenicprotein or an immunogenic polypeptide thereof encoded by the firstpolynucleotide is derived from a filovirus. In yet another embodiment,the antigenic proteins comprise the amino acid sequences selected fromthe group of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, andSEQ ID NO: 5. Most preferably, the MVA vector comprises a polynucleotideencoding the antigenic proteins having the amino acid sequences of SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5.

More preferably, the adenovirus vector comprises a polynucleotideencoding at least one antigenic protein having the amino acid sequenceof SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3. In a more preferredembodiment, the adenovirus vector comprises a polynucleotide encodingthe antigenic protein having the amino acid sequence of SEQ ID NO: 1.Preferably said adenovirus vector is an rAd26 vector.

The invention further relates to:

-   -   a. a first composition comprising an immunologically effective        amount of a MVA vector comprising a first polynucleotide        encoding an antigenic protein or an immunogenic polypeptide        thereof; and    -   b. a second composition comprising an immunologically effective        amount of an adenovirus vector comprising a second        polynucleotide encoding the antigenic protein or an immunogenic        polypeptide thereof for boosting the immune response;    -   wherein the first composition is administered to a human subject        for priming the immune response, and the second composition is        administered to the human subject for boosting the immune        response, for use in inducing an enhanced humoral and/or        cellular immune response in the human subject relative to the        humoral and/or cellular immune response that would be observed        if the second composition would be administered for priming and        the first composition would be administered for boosting the        immune response.

In one embodiment of the invention, the enhanced immune responsegenerated by said compositions a. and b. comprises an increase of theantibody response against the antigenic protein in the human subjectcombined with a CD4+ and CD8+ response [e.g., a response characterizedby the presence of a high proportion of CD4+ and CD8+ responders, suchas more than 50%, 60%, 70%, 80%, 90% or 100% of subjects tested asdetermined by an ICS assay, with a median total cytokine response ofabout 0.2%, 0.3%, 0.4%, 0.5% or more]. In another embodiment of theinvention, the enhanced CD4+ and CD8+ T cell responses generated by saidcompositions a. and b. comprises an increase or induction ofpolyfunctional CD4+ and CD8+ T cells specific to the antigenic protein.Such polyfunctional CD4+ T cells express more than one cytokine, such astwo or more of IFN-gamma, IL-2 and TNF-alpha.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing summary, as well as the following detailed description ofthe invention, will be better understood when read in conjunction withthe appended drawings. It should be understood that the invention is notlimited to the precise embodiments shown in the drawings.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

In the drawings:

FIG. 1 summarizes the grouping in an animal study;

FIG. 2 illustrates the experimental design of the study;

FIG. 3 shows the outcome of challenge with the challenge strain EbolaZaire Kikwit 1995;

FIG. 4 shows the Ebola Zaire glycoprotein specific humoral immuneresponse (assessed by ELISA) observed from the animal study: very highantibody titers were obtained independently of the vaccination regimes(ND=time-point not analyzed);

FIG. 5 shows the Sudan Gulu glycoprotein specific humoral immuneresponse (assessed by ELISA) observed from the animal study: very highantibody titers were obtained independently of the vaccination regimes(ND=time-point not analyzed);

FIG. 6 shows the Marburg Angola glycoprotein specific humoral immuneresponse (assessed by ELISA) observed from the animal study: very highantibody titers were obtained independently of the vaccination regimes(ND=time-point not analyzed);

FIG. 7 shows the specific cellular immune response to ZEBOV, SEBOV andMARVA GP analyzed by an IFN-γ ELISPOT;

FIG. 8 shows the specific immune response to ZEBOV GP analyzed by ananti-EBOV GP ELISA, wherein at 21 days post boost immunization, a higherhumoral immune response post boost immunization is observed in subjectsimmunized with MVA as a prime and Ad26 as a boost than with the reverseorder of vaccines;

FIG. 9 shows the specific T cell response to ZEBOV GP in humans analyzedby ELISpot assay;

FIG. 10 shows the specific CD8+ cellular immune response to ZEBOV GP inhumans analyzed by ICS assay;

FIG. 11 shows the functionality of the EBOV GP-specific CD8+ T cellresponses in humans by ICS assay when using a 28 days prime boostinterval;

FIG. 12 shows the functionality of the EBOV GP-specific CD8+ T cellresponses in humans by ICS assay when using a 56 days prime boostinterval;

FIG. 13 shows the specific CD4+ cellular immune response to ZEBOV GP inhumans analyzed by ICS assay; and

FIG. 14 shows the functionality of the EBOV GP-specific CD4+ T cellresponses in humans by ICS assay when using a 28 days prime boostinterval;

FIG. 15 shows the functionality of the EBOV GP-specific CD4+ T cellresponses in humans by ICS assay when using a 56 days prime boostinterval;

FIG. 16 shows the immune response induced by a prime immunization withAd26.ZEBOV followed by a MVA-BN-Filo boost 14 days later assessed byELISA (A), ELIspot (B), and ICS (C and D);

FIG. 17 shows the specific immune response to ZEBOV GP analyzed by ananti-EBOV GP ELISA;

FIG. 18 shows the specific T cell response to ZEBOV GP in humansanalyzed by ELISpot assay;

FIG. 19 shows the strong and balanced CD4+ (A) and CD8+ (B) cellularimmune response specific to ZEBOV GP in humans analyzed by ICS assay andthe functionality of the EBOV GP-specific CD8+ (C) and CD4+ (D) T cellresponses in humans by ICS assay when using MVA as a prime and Ad26 as aboost 14 days later.

FIG. 20 shows EBOV Mayinga GP-binding Antibodies Elicited by VaccinationWith Ad26.ZEBOV/MVA-BN-Filo and MVA-BN-Filo/Ad26.ZEBOV RegimensDetermined by GP ELISA. Vaccination regimens are indicated below x-axis.High IM and standard IM refer to dose and route of MVA BN Filo.Horizontal dotted line indicates LOD.

DETAILED DESCRIPTION OF THE INVENTION

Various publications, articles and patents are cited or described in thebackground and throughout the specification; each of these references isherein incorporated by reference in its entirety. Discussion ofdocuments, acts, materials, devices, articles or the like which has beenincluded in the present specification is for the purpose of providingcontext for the invention. Such discussion is not an admission that anyor all of these matters form part of the prior art with respect to anyinventions disclosed or claimed.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention pertains. Otherwise, certain terms usedherein have the meanings as set forth in the specification. All patents,published patent applications and publications cited herein areincorporated by reference as if set forth fully herein. It must be notedthat as used herein and in the appended claims, the singular forms “a,”“an,” and “the” include plural reference unless the context clearlydictates otherwise.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the invention.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integer or step. Whenused herein the term “comprising” can be substituted with the term“containing” or “including” or sometimes when used herein with the term“having”. When used herein “consisting of” excludes any element, step,or ingredient not specified in the claim element. When used herein,“consisting essentially of” does not exclude materials or steps that donot materially affect the basic and novel characteristics of the claim.In each instance herein any of the terms “comprising”, “consistingessentially of” and “consisting of” can be replaced with either of theother two terms.

As used herein, the conjunctive term “and/or” between multiple recitedelements is understood as encompassing both individual and combinedoptions. For instance, where two elements are conjoined by “and/or”, afirst option refers to the applicability of the first element withoutthe second. A second option refers to the applicability of the secondelement without the first. A third option refers to the applicability ofthe first and second elements together. Any one of these options isunderstood to fall within the meaning, and therefore satisfy therequirement of the term “and/or” as used herein. Concurrentapplicability of more than one of the options is also understood to fallwithin the meaning, and therefore satisfy the requirement of the term“and/or.”

As used herein, “subject” means any animal, preferably a mammal, mostpreferably a human, to whom will be or has been treated by a methodaccording to an embodiment of the invention. The term “mammal” as usedherein, encompasses any mammal. Examples of mammals include, but are notlimited to, cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits,guinea pigs, monkeys, humans, etc., more preferably a human.

As used herein, the term “protective immunity” or “protective immuneresponse” means that the vaccinated subject is able to control aninfection or a disease related to an antigenic protein or immunogenicpolypeptide thereof against which the vaccination was done. Usually, thesubject having developed a “protective immune response” develops onlymild to moderate clinical symptoms or no symptoms at all. Usually, asubject having a “protective immune response” or “protective immunity”against a certain antigenic protein will not die as a result of aninfection or disease related to the antigenic protein.

The antigenic protein can be a native protein from a pathogen or atumor, or a modified protein based on a native protein from a pathogenor a tumor.

As used herein, the term “pathogen” refers to an infectious agent suchas a virus, a bacterium, a fungus, a parasite, or a prion that causesdisease in its host.

As used herein, the term “enhanced” when used with respect to an immuneresponse, such as a CD4+ T cell response, an antibody response, or aCD8+ T cell response, refers to an increase in the immune response in ahuman subject administered with a prime-boost combination of replicationincompetent MVA and adenovirus vectors according to the invention,relative to the corresponding immune response observed from the humansubject administered with a reverse prime-boost combination, wherein theadenovirus vector is provided as a prime and the MVA vector is providedto boost the immune response, using the same prime-boost interval.

As used herein, the term “dominant CD4+ or CD8+T cell response” refersto a T cell immune response that is characterized by observing highproportion of immunogen-specific CD4+ T cells within the population oftotal responding T cells following vaccination. The totalimmunogen-specific T-cell response can be determined by an IFN-gammaELISPOT assay. The immunogen-specific CD4+ or CD8+ T cell immuneresponse can be determined by an ICS assay. For example, a dominant CD4+T cell response can comprise an antigen specific CD4+ T cell responsethat is more than 50%, such as 51%, 60%, 70%, 80%, 90% or 100% of thetotal antigen specific T-cell responses in the human subject.Preferably, the dominant CD4+ T cell response also represents 0.1% ormore, such as 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, or more of the totalcytokine responses in the human subject.

As used herein, the term “enhanced antibody response” refers to anantibody response in a human subject administered with a prime-boostcombination of replication incompetent MVA and adenovirus vectorsaccording to the invention, that is increased by a factor of at least1.5, 2, 2.5, or more relative to the corresponding immune responseobserved from the human subject administered with a reverse prime-boostcombination, wherein the adenovirus vector is provided as a prime andthe MVA vector is provided to boost the immune response, using the sameprime-boost interval.

As used herein, the term “polyfunctional” when used with respect to CD4+or CD8+ T cells means T cells that express more than one cytokine, suchas at least two of: IL-2, IFN-gamma, and TNF-alpha.

An “adenovirus capsid protein” refers to a protein on the capsid of anadenovirus (e.g., Ad 26 or Ad 35) that is involved in determining theserotype and/or tropism of a particular adenovirus. Adenoviral capsidproteins typically include the fiber, penton and/or hexon proteins. Asused herein a “Ad26 capsid protein” or a “Ad35 capsid protein” can be,for example, a chimeric capsid protein that includes at least a part ofan Ad26 or Ad35 capsid protein. In certain embodiments, the capsidprotein is an entire capsid protein of Ad26 or of Ad35. In certainembodiments, the hexon, penton and fiber are of Ad26 or of Ad35.

The terms “adjuvant” and “immune stimulant” are used interchangeablyherein, and are defined as one or more substances that cause stimulationof the immune system. In this context, an adjuvant is used to enhance animmune response to the adenovirus and/or MVA vectors of the invention.

The term “corresponding to”, when applied to positions of amino acidresidues in sequences, means corresponding positions in a plurality ofsequences when the sequences are optimally aligned.

The terms “identical” or percent “identity,” in the context of two ormore nucleic acids or polypeptide sequences, (e.g., glycoproteins offilovirus and polynucleotides that encode them) refer to two or moresequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same, whencompared and aligned for maximum correspondence, as measured using oneof the following sequence comparison algorithms or by visual inspection.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

Optimal alignment of sequences for comparison can be conducted, e.g., bythe local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482(1981), by the homology alignment algorithm of Needleman & Wunsch, J.Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson& Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by visual inspection (see generally,Current Protocols in Molecular Biology, F. M. Ausubel et al., eds.,Current Protocols, a joint venture between Greene Publishing Associates,Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)).

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and BLAST 2.0algorithms, which are described in Altschul et al. (1990) J. Mol. Biol.215: 403-410 and Altschuel et al. (1977) Nucleic Acids Res. 25:3389-3402, respectively. Software for performing BLAST analyses ispublicly available through the National Center for BiotechnologyInformation. This algorithm involves first identifying high scoringsequence pairs (HSPs) by identifying short words of length W in thequery sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al, supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.

Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always >0)and N (penalty score for mismatching residues; always <0). For aminoacid sequences, a scoring matrix is used to calculate the cumulativescore. Extension of the word hits in each direction are halted when: thecumulative alignment score falls off by the quantity X from its maximumachieved value; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTNprogram (for nucleotide sequences) uses as defaults a wordlength (W) of11, an expectation (E) of 10, M=5, N=−4, and a comparison of bothstrands. For amino acid sequences, the BLASTP program uses as defaults awordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915(1989)).

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA90:5873-5787 (1993)). One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a nucleic acidis considered similar to a reference sequence if the smallest sumprobability in a comparison of the test nucleic acid to the referencenucleic acid is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

A further indication that two nucleic acid sequences or polypeptides aresubstantially identical is that the polypeptide encoded by the firstnucleic acid is immunologically cross reactive with the polypeptideencoded by the second nucleic acid, as described below. Thus, apolypeptide is typically substantially identical to a secondpolypeptide, for example, where the two peptides differ only byconservative substitutions. Another indication that two nucleic acidsequences are substantially identical is that the two moleculeshybridize to each other under stringent conditions, as described below.

The term “substantially similar” in the context of the filovirusantigenic proteins of the invention indicates that a polypeptidecomprises a sequence with at least 90%, preferably at least 95% sequenceidentity to the reference sequence over a comparison window of 10-20amino acids. Percentage of sequence identity is determined by comparingtwo optimally aligned sequences over a comparison window, wherein theportion of the polynucleotide sequence in the comparison window maycomprise additions or deletions (i.e., gaps) as compared to thereference sequence (which does not comprise additions or deletions) foroptimal alignment of the two sequences. The percentage is calculated bydetermining the number of positions at which the identical nucleic acidbase or amino acid residue occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison and multiplyingthe result by 100 to yield the percentage of sequence identity.

It is discovered in the invention that heterologous prime-boostcombinations, in particular, MVA priming followed by Ad26 boosting, aresurprisingly effective in generating protective immune responses inhuman subjects.

Antigenic Proteins

Any DNA of interest can be inserted into the viral vectors describedherein to be expressed heterologously from the vectors. Foreign genesfor insertion into the genome of a virus in expressible form can beobtained using conventional techniques for isolating a desired gene. Fororganisms which contain a DNA genome, the genes encoding an antigen ofinterest can be isolated from the genomic DNA; for organisms with RNAgenomes, the desired gene can be isolated from cDNA copies of thegenome. The antigenic protein can also be encoded by a recombinant DNAthat is modified based on a naturally occurring sequence, e.g., tooptimize the antigenic response, gene expression, etc.

In certain embodiments of the invention, MVA-prime and adenovirus-boostcombinations of replication incompetent vectors generate an enhancedimmune response to an antigenic protein or an immunogenic polypeptidethereof in a human subject. The antigenic protein can be any antigenicprotein related to an infection or disease.

According to embodiments of the invention, the antigenic protein orimmunogenic polypeptide thereof can be isolated from, or derived from, apathogen, such as a virus (e.g., Filovirus, adenovirus, arbovirus,astrovirus, coronavirus, coxsackie virus, cytomegalovirus, Dengue virus,Epstein-Barr virus, hepatitis virus, herpesvirus, human immunodeficiencyvirus, human papilloma virus, human T-lymphotropic virus, influenzavirus, JC virus, lymphocytic choriomeningitis virus, measles virus,molluscum contagiosum virus, mumps virus, norovirus, parovirus,poliovirus, rabies virus, respiratory syncytial virus, rhinovirus,rotavirus, rotavirus, rubella virus, smallpox virus, varicella zostervirus, West Nile virus, etc.), a bacteria (e.g., Campylobacter jejuni,Escherichia coli, Helicobacter pylori, Mycobacterium tuberculosis,Neisseria gonorrhoeae, Neisseria meningitides, Salmonella, Shigella,Staphylococcus aureus, Streptococcus, etc.), a fungus (e.g.,Coccidioides immitis, Blastomyces dermatitidis, Cryptococcus neoformans,Candida species, Aspergillus species, etc.), a protozoan (e.g.,Plasmodium, Leishmania, Trypanosome, cryptosporidiums, isospora,Naegleria fowleri, Acanthamoeba, Balamuthia mandrillaris, Toxoplasmagondii, Pneumocystis carinii, etc.), or a cancer (e.g., bladder cancer,breast cancer, colon and rectal cancer, endometrial cancer, kidneycancer, leukemia, lung cancer, melanoma, non-Hodgkin lymphoma,pancreatic cancer, prostate cancer, thyroid cancer, etc.).

In some embodiments, nucleic acids express antigenic domains rather thanthe entire antigenic protein. These fragments can be of any lengthsufficient to be immunogenic or antigenic. Fragments can be at leastfour amino acids long, preferably 8-20 amino acids, but can be longer,such as, e.g., 100, 200, 660, 800, 1000, 1200, 1600, 2000 amino acidslong or more, or any length in between.

In some embodiments, at least one nucleic acid fragment encoding anantigenic protein or immunogenic polypeptide thereof is inserted into aviral vector. In another embodiment, about 2-8 different nucleic acidsencoding different antigenic proteins are inserted into one or more ofthe viral vectors. In some embodiments, multiple immunogenic fragmentsor subunits of various proteins can be used. For example, severaldifferent epitopes from different sites of a single protein or fromdifferent proteins of the same species, or from a protein ortholog fromdifferent species can be expressed from the vectors.

Filovirus Antigenic Proteins

The Ebola viruses, and the genetically-related Marburg virus, arefiloviruses associated with outbreaks of highly lethal hemorrhagic feverin humans and primates in North America, Europe, and Africa (Peters, C.J. et al. in: Fields Virology, eds. Fields, B. N. et al. 1161-1176,Philadelphia, Lippincott-Raven, 1996; Peters, C. J. et al. 1994 SeminVirol 5:147-154). Although several subtypes have been defined, thegenetic organization of these viruses is similar, each containing sevenlinearly arrayed genes. Among the viral proteins, the envelopeglycoprotein exists in two alternative forms, a 50-70 kilodalton (kDa)secreted protein (sGP) and a 130 kDa transmembrane glycoprotein (GP)generated by RNA editing that mediates viral entry (Peters, C. J. et al.in: Fields Virology, eds. Fields, B. N. et al. 1161-1176, Philadelphia,Lippincott-Raven, 1996; Sanchez, A. et al. 1996 PNAS USA 93:3602-3607).Other structural gene products include the nucleoprotein (NP), matrixproteins VP24 and VP40, presumed nonstructural proteins VP30 and VP35,and the viral polymerase (reviewed in Peters, C. J. et al. in: FieldsVirology, eds. Fields, B. N. et al. 1161-1176, Philadelphia,Lippincott-Raven, 1996).

The nucleic acid molecules comprised in the adenovirus and MVA vectorsmay encode structural gene products of any filovirus species, such assubtypes of Zaire (type species, also referred to herein as ZEBOV),Sudan (also referred to herein as SEBOV), Reston, Bundibugyo, and IvoryCoast. There is a single species of Marburg virus (also referred toherein as MARV).

The adenoviral vectors and MVA vectors of the invention can be used toexpress antigenic proteins which are proteins comprising an antigenicdeterminant of a wide variety of filovirus antigens. In a typical andpreferred embodiment, the vectors of the invention include nucleic acidencoding the transmembrane form of the viral glycoprotein (GP). In otherembodiments, the vectors of the invention may encode the secreted formof the viral glycoprotein (ssGP), or the viral nucleoprotein (NP).

One of skill will recognize that the nucleic acid molecules encoding thefilovirus antigenic protein can be modified, e.g., the nucleic acidmolecules set forth herein can be mutated, as long as the modifiedexpressed protein elicits an immune response against a pathogen ordisease. Thus, as used herein, the term “antigenic protein” or“filovirus protein” refers to a protein that comprises at least oneantigenic determinant of a filovirus protein described above. The termencompasses filovirus glycoproteins (i.e., gene products of a filovirus)or filovirus nucleoprotein as well as recombinant proteins that compriseone or more filovirus glycoprotein determinants. The term antigenicproteins also encompasses antigenic proteins that are substantiallysimilar.

In some embodiments, the protein can be mutated so that it is less toxicto cells (see e.g., WO/2006/037038) or can be expressed with increasedor decreased level in the cells. The invention also includes vaccinescomprising a combination of nucleic acid molecules. For example, andwithout limitation, nucleic acid molecules encoding GP, ssGP and NP ofthe Zaire, Sudan, Marburg and Ivory Coast/Taï Forest Ebola strains canbe combined in any combination, in one vaccine composition.

Adenoviruses

An adenovirus according to the invention belongs to the family of theAdenoviridae and preferably is one that belongs to the genusMastadenovirus. It can be a human adenovirus, but also an adenovirusthat infects other species, including but not limited to a bovineadenovirus (e.g. bovine adenovirus 3, BAdV3), a canine adenovirus (e.g.CAdV2), a porcine adenovirus (e.g. PAdV3 or 5), or a simian adenovirus(which includes a monkey adenovirus and an ape adenovirus, such as achimpanzee adenovirus or a gorilla adenovirus). Preferably, theadenovirus is a human adenovirus (HAdV, or AdHu; in the invention ahuman adenovirus is meant if referred to Ad without indication ofspecies, e.g. the brief notation “Ad5” means the same as HAdV5, which ishuman adenovirus serotype 5), or a simian adenovirus such as chimpanzeeor gorilla adenovirus (ChAd, AdCh, or SAdV).

Most advanced studies have been performed using human adenoviruses, andhuman adenoviruses are preferred according to certain aspects of theinvention. In certain preferred embodiments, the recombinant adenovirusaccording to the invention is based upon a human adenovirus. Inpreferred embodiments, the recombinant adenovirus is based upon a humanadenovirus serotype 5, 11, 26, 34, 35, 48, 49 or 50. According to aparticularly preferred embodiment of the invention, an adenovirus is ahuman adenovirus of one of the serotypes 26 or 35.

An advantage of these serotypes is a low seroprevalence and/or lowpre-existing neutralizing antibody titers in the human population.Preparation of rAd26 vectors is described, for example, in WO2007/104792 and in Abbink et al., (2007) Virol 81(9): 4654-63, both ofwhich are incorporated by reference herein in their entirety. Exemplarygenome sequences of Ad26 are found in GenBank Accession EF 153474 and inSEQ ID NO:1 of WO 2007/104792. Preparation of rAd35 vectors isdescribed, for example, in U.S. Pat. No. 7,270,811, in WO 00/70071, andin Vogels et al., (2003) J Virol 77(15): 8263-71, all of which areincorporated by reference herein in their entirety. Exemplary genomesequences of Ad35 are found in GenBank Accession AC_000019 and in FIG. 6of WO 00/70071.

Simian adenoviruses generally also have a low seroprevalence and/or lowpre-existing neutralizing antibody titers in the human population, and asignificant amount of work has been reported using chimpanzee adenovirusvectors (e.g. U.S. Pat. No. 6,083,716; WO 2005/071093; WO 2010/086189;WO 2010085984; Farina et al, 2001, J Virol 75: 11603-13; Cohen et al,2002, J Gen Virol 83: 151-55; Kobinger et al, 2006, Virology 346:394-401; Tatsis et al., 2007, Molecular Therapy 15: 608-17; see alsoreview by Bangari and Mittal, 2006, Vaccine 24: 849-62; and review byLasaro and Ertl, 2009, Mol Ther 17: 1333-39). Hence, in other preferredembodiments, the recombinant adenovirus according to the invention isbased upon a simian adenovirus, e.g. a chimpanzee adenovirus. In certainembodiments, the recombinant adenovirus is based upon simian adenovirustype 1, 7, 8, 21, 22, 23, 24, 25, 26, 27.1, 28.1, 29, 30, 31.1, 32, 33,34, 35.1, 36, 37.2, 39, 40.1, 41.1, 42.1, 43, 44, 45, 46, 48, 49, 50 orSA7P.

Adenoviral Vectors rAd26 and rAd35

In a preferred embodiment according to the invention the adenoviralvectors comprise capsid proteins from two rare serotypes: Ad26 and Ad35.In the typical embodiment, the vector is an rAd26 or rAd35 virus.

Thus, the vectors that can be used in the invention comprise an Ad26 orAd35 capsid protein (e.g., a fiber, penton or hexon protein). One ofskill will recognize that it is not necessary that an entire Ad26 orAd35 capsid protein be used in the vectors of the invention. Thus,chimeric capsid proteins that include at least a part of an Ad26 or Ad35capsid protein can be used in the vectors of the invention. The vectorsof the invention may also comprise capsid proteins in which the fiber,penton, and hexon proteins are each derived from a different serotype,so long as at least one capsid protein is derived from Ad26 or Ad35. Inpreferred embodiments, the fiber, penton and hexon proteins are eachderived from Ad26 or each from Ad35.

One of skill will recognize that elements derived from multipleserotypes can be combined in a single recombinant adenovirus vector.Thus, a chimeric adenovirus that combines desirable properties fromdifferent serotypes can be produced. Thus, in some embodiments, achimeric adenovirus of the invention could combine the absence ofpre-existing immunity of the Ad26 and Ad35 serotypes withcharacteristics such as temperature stability, assembly, anchoring,production yield, redirected or improved infection, stability of the DNAin the target cell, and the like.

In certain embodiments the recombinant adenovirus vector useful in theinvention is derived mainly or entirely from Ad35 or from Ad26 (i.e.,the vector is rAd35 or rAd26). In some embodiments, the adenovirus isreplication deficient, e.g. because it contains a deletion in the E1region of the genome. For the adenoviruses of the invention, beingderived from Ad26 or Ad35, it is typical to exchange the E4-orf6 codingsequence of the adenovirus with the E4-orf6 of an adenovirus of humansubgroup C such as Ad5. This allows propagation of such adenoviruses inwell-known complementing cell lines that express the E1 genes of Ad5,such as for example 293 cells, PER.C6 cells, and the like (see, e.g.Havenga et al, 2006, J Gen Virol 87: 2135-43; WO 03/104467). In certainembodiments, the adenovirus is a human adenovirus of serotype 35, with adeletion in the E1 region into which the nucleic acid encoding theantigen has been cloned, and with an E4 orf6 region of Ad5. In certainembodiments, the adenovirus is a human adenovirus of serotype 26, with adeletion in the E1 region into which the nucleic acid encoding theantigen has been cloned, and with an E4 orf6 region of Ad5. For the Ad35adenovirus, it is typical to retain the 3′ end of the E1B 55K openreading frame in the adenovirus, for instance the 166 bp directlyupstream of the pIX open reading frame or a fragment comprising thissuch as a 243 bp fragment directly upstream of the pIX start codon,marked at the 5′ end by a Bsu361 restriction site, since this increasesthe stability of the adenovirus because the promoter of the pIX gene ispartly residing in this area (see, e.g. Havenga et al, 2006, supra; WO2004/001032).

The preparation of recombinant adenoviral vectors is well known in theart.

Preparation of rAd26 vectors is described, for example, in WO2007/104792 and in Abbink et al., (2007) Virol 81(9): 4654-63. Exemplarygenome sequences of Ad26 are found in GenBank Accession EF 153474 and inSEQ ID NO:1 of WO 2007/104792. Preparation of rAd35 vectors isdescribed, for example, in U.S. Pat. No. 7,270,811 and in Vogels et al.,(2003) J Virol 77(15): 8263-71. An exemplary genome sequence of Ad35 isfound in GenBank Accession AC_000019.

In an embodiment of the invention, the vectors useful for the inventioninclude those described in WO2012/082918, the disclosure of which isincorporated herein by reference in its entirety.

Typically, a vector useful in the invention is produced using a nucleicacid comprising the entire recombinant adenoviral genome (e.g., aplasmid, cosmid, or baculovirus vector). Thus, the invention alsoprovides isolated nucleic acid molecules that encode the adenoviralvectors of the invention. The nucleic acid molecules of the inventioncan be in the form of RNA or in the form of DNA obtained by cloning orproduced synthetically. The DNA can be double-stranded orsingle-stranded.

The adenovirus vectors useful the invention are typically replicationdefective. In these embodiments, the virus is renderedreplication-defective by deletion or inactivation of regions critical toreplication of the virus, such as the E1 region. The regions can besubstantially deleted or inactivated by, for example, inserting the geneof interest (usually linked to a promoter). In some embodiments, thevectors of the invention may contain deletions in other regions, such asthe E2, E3 or E4 regions or insertions of heterologous genes linked to apromoter. For E2- and/or E4-mutated adenoviruses, generally E2- and/orE4-complementing cell lines are used to generate recombinantadenoviruses. Mutations in the E3 region of the adenovirus need not becomplemented by the cell line, since E3 is not required for replication.

A packaging cell line is typically used to produce sufficient amount ofadenovirus vectors of the invention. A packaging cell is a cell thatcomprises those genes that have been deleted or inactivated in areplication-defective vector, thus allowing the virus to replicate inthe cell.

Suitable cell lines include, for example, PER.C6, 911, 293, and El A549.

In some embodiments, the Adenovirus virus may express genes or portionsof genes that encode antigenic peptides. These foreign, heterologous orexogenous peptides or polypeptides can include sequences that areimmunogenic such as, for example, tumor-specific antigens (TSAs),bacterial, viral, fungal, and protozoal antigens.

As noted above, a wide variety of filovirus glycoproteins can beexpressed in the vectors. If required, the heterologous gene encodingthe filovirus glycoproteins can be codon-optimized to ensure properexpression in the treated host (e.g., human). Codon-optimization is atechnology widely applied in the art. Typically, the heterologous geneis cloned into the E1 and/or the E3 region of the adenoviral genome.

The heterologous filovirus gene can be under the control of (i.e.,operably linked to) an adenovirus-derived promoter (e.g., the Major LatePromoter) or can be under the control of a heterologous promoter.Examples of suitable heterologous promoters include the CMV promoter andthe RSV promoter. Preferably, the promoter is located upstream of theheterologous gene of interest within an expression cassette.

In a preferred embodiment of the invention, the adenovirus vectorsuseful for the invention can comprise a wide variety of filovirusglycoproteins known to those of skill in the art. In a further preferredembodiment of the invention, the rAd vector(s) comprises one or more GPsselected from the group consisting of GPs of Zaire ebolavirus (EBOV),GPs of Sudan ebolavirus (SUDV), GPs of Marburg virus (MARV), and GPssubstantially similar thereto.

MVA Vectors

MVA vectors useful for the invention utilize attenuated virus derivedfrom Modified Vaccinia Ankara virus which is characterized by the lossof their capabilities to reproductively replicate in human cell lines.

In some embodiments, the MVA virus may express genes or portions ofgenes that encode antigenic peptides. These foreign, heterologous orexogenous peptides or polypeptides can include sequences that areimmunogenic such as, for example, tumor-specific antigens (TSAs),bacterial, viral, fungal, and protozoal antigens.

In other embodiments, the MVA vectors express a wide variety offilovirus glycoproteins as well as other structural filovirus proteins,such as VP40 and nucleoprotein (NP). In one aspect, the inventionprovides a recombinant modified vaccinia virus Ankara (MVA) comprising anucleotide sequence encoding an antigenic determinant of a filovirusglycoprotein (GP), in particular an envelope glycoprotein. In anotheraspect, the invention provides a recombinant MVA vector comprising aheterologous nucleotide sequence encoding an antigenic determinant of aFilovirus glycoprotein, in particular an envelope glycoprotein, and aheterologous nucleotide sequence encoding an antigenic determinant of afurther Filovirus protein.

MVA has been generated by more than 570 serial passages on chickenembryo fibroblasts of the dermal vaccinia strain Ankara [Chorioallantoisvaccinia virus Ankara virus, CVA; for review see Mayr et al. (1975),Infection 3, 6-14] that was maintained in the Vaccination Institute,Ankara, Turkey for many years and used as the basis for vaccination ofhumans. However, due to the often severe post-vaccination complicationsassociated with vaccinia viruses, there were several attempts togenerate a more attenuated, safer smallpox vaccine.

During the period of 1960 to 1974, Prof. Anton Mayr succeeded inattenuating CVA by over 570 continuous passages in CEF cells [Mayr etal. (1975)]. It was shown in a variety of animal models that theresulting MVA was avirulent [Mayr, A. & Danner, K. (1978), Dev. Biol.Stand. 41: 225-234]. As part of the early development of MVA as apre-smallpox vaccine, there were clinical trials using MVA-517 incombination with Lister Elstree [Stickl (1974), Prev. Med. 3: 97-101;Stickl and Hochstein-Mintzel (1971), Munch. Med. Wochenschr. 113:1149-1153] in subjects at risk for adverse reactions from vaccinia. In1976, MVA derived from MVA-571 seed stock (corresponding to the 571stpassage) was registered in Germany as the primer vaccine in a two-stageparenteral smallpox vaccination program. Subsequently, MVA-572 was usedin approximately 120,000 Caucasian individuals, the majority childrenbetween 1 and 3 years of age, with no reported severe side effects, eventhough many of the subjects were among the population with high risk ofcomplications associated with vaccinia (Mayr et al. (1978), Zentralbl.Bacteriol. (B) 167:375-390). MVA-572 was deposited at the EuropeanCollection of Animal Cell Cultures as ECACC V94012707.

As a result of the passaging used to attenuate MVA, there are a numberof different strains or isolates, depending on the number of passagesconducted in CEF cells. For example, MVA-572 was used in a small dose asa pre-vaccine in Germany during the smallpox eradication program, andMVA-575 was extensively used as a veterinary vaccine. MVA as well asMVA-BN lacks approximately 15% (31 kb from six regions) of the genomecompared with ancestral CVA virus. The deletions affect a number ofvirulence and host range genes, as well as the gene for Type A inclusionbodies. MVA-575 was deposited on Dec. 7, 2000, at the EuropeanCollection of Animal Cell Cultures (ECACC) under Accession No.V00120707. The attenuated CVA-virus MVA (Modified Vaccinia Virus Ankara)was obtained by serial propagation (more than 570 passages) of the CVAon primary chicken embryo fibroblasts.

Even though Mayr et al. demonstrated during the 1970s that MVA is highlyattenuated and avirulent in humans and mammals, certain investigatorshave reported that MVA is not fully attenuated in mammalian and humancell lines since residual replication might occur in these cells[Blanchard et al. (1998), J. Gen. Virol. 79:1159-1167; Carroll & Moss(1997), Virology 238:198-211; U.S. Pat. No. 5,185,146; Ambrosini et al.(1999), J. Neurosci. Res. 55: 569]. It is assumed that the resultsreported in these publications have been obtained with various knownstrains of MVA, since the viruses used essentially differ in theirproperties, particularly in their growth behavior in various cell lines.Such residual replication is undesirable for various reasons, includingsafety concerns in connection with use in humans.

Strains of MVA having enhanced safety profiles for the development ofsafer products, such as vaccines or pharmaceuticals, have been developedby Bavarian Nordic. MVA was further passaged by Bavarian Nordic and isdesignated MVA-BN, a representative sample of which was deposited onAug. 30, 2000 at the European Collection of Cell Cultures (ECACC) underAccession No. V00083008. MVA-BN is further described in WO 02/42480 (US2003/0206926) and WO 03/048184 (US 2006/0159699), both of which areincorporated by reference herein in their entirety.

MVA-BN can attach to and enter human cells where virally-encoded genesare expressed very efficiently. MVA-BN is strongly adapted to primarychicken embryo fibroblast (CEF) cells and does not replicate in humancells. In human cells, viral genes are expressed, and no infectiousvirus is produced. MVA-BN is classified as Biosafety Level 1 organismaccording to the Centers for Disease Control and Prevention in theUnited States. Preparations of MVA-BN and derivatives have beenadministered to many types of animals, and to more than 2000 humansubjects, including immune-deficient individuals. All vaccinations haveproven to be generally safe and well tolerated. Despite its highattenuation and reduced virulence, in preclinical studies MVA-BN hasbeen shown to elicit both humoral and cellular immune responses tovaccinia and to heterologous gene products encoded by genes cloned intothe MVA genome [E. Harrer et al. (2005), Antivir. Ther. 10(2):285-300;A. Cosma et al. (2003), Vaccine 22(1):21-9; M. Di Nicola et al. (2003),Hum. Gene Ther. 14(14):1347-1360; M. Di Nicola et al. (2004), Clin.Cancer Res., 10(16):5381-5390].

“Derivatives” or “variants” of MVA refer to viruses exhibitingessentially the same replication characteristics as MVA as describedherein, but exhibiting differences in one or more parts of theirgenomes. MVA-BN as well as a derivative or variant of MVA-BN fails toreproductively replicate in vivo in humans and mice, even in severelyimmune suppressed mice. More specifically, MVA-BN or a derivative orvariant of MVA-BN has preferably also the capability of reproductivereplication in chicken embryo fibroblasts (CEF), but no capability ofreproductive replication in the human keratinocyte cell line HaCat[Boukamp et al (1988), J. Cell Biol. 106: 761-771], the human boneosteosarcoma cell line 143B (ECACC Deposit No. 91112502), the humanembryo kidney cell line 293 (ECACC Deposit No. 85120602), and the humancervix adenocarcinoma cell line HeLa (ATCC Deposit No. CCL-2).Additionally, a derivative or variant of MVA-BN has a virusamplification ratio at least two fold less, more preferably three-foldless than MVA-575 in Hela cells and HaCaT cell lines. Tests and assayfor these properties of MVA variants are described in WO 02/42480 (US2003/0206926) and WO 03/048184 (US 2006/0159699).

The term “not capable of reproductive replication” or “no capability ofreproductive replication” is, for example, described in WO 02/42480,which also teaches how to obtain MVA having the desired properties asmentioned above. The term applies to a virus that has a virusamplification ratio at 4 days after infection of less than 1 using theassays described in WO 02/42480 or in U.S. Pat. No. 6,761,893, both ofwhich are incorporated by reference herein in their entirety.

The term “fails to reproductively replicate” refers to a virus that hasa virus amplification ratio at 4 days after infection of less than 1.Assays described in WO 02/42480 or in U.S. Pat. No. 6,761,893 areapplicable for the determination of the virus amplification ratio.

The amplification or replication of a virus is normally expressed as theratio of virus produced from an infected cell (output) to the amountoriginally used to infect the cell in the first place (input) referredto as the “amplification ratio”. An amplification ratio of “1” definesan amplification status where the amount of virus produced from theinfected cells is the same as the amount initially used to infect thecells, meaning that the infected cells are permissive for virusinfection and reproduction. In contrast, an amplification ratio of lessthan 1, i.e., a decrease in output compared to the input level,indicates a lack of reproductive replication and therefore attenuationof the virus.

The advantages of MVA-based vaccine include their safety profile as wellas availability for large scale vaccine production. Preclinical testshave revealed that MVA-BN demonstrates superior attenuation and efficacycompared to other MVA strains (WO 02/42480). An additional property ofMVA-BN strains is the ability to induce substantially the same level ofimmunity in vaccinia virus prime/vaccinia virus boost regimes whencompared to DNA-prime/vaccinia virus boost regimes.

The recombinant MVA-BN viruses, the most preferred embodiment herein,are considered to be safe because of their distinct replicationdeficiency in mammalian cells and their well-established avirulence.Furthermore, in addition to its efficacy, the feasibility of industrialscale manufacturing can be beneficial. Additionally, MVA-based vaccinescan deliver multiple heterologous antigens and allow for simultaneousinduction of humoral and cellular immunity.

MVA vectors useful for the invention can be prepared using methods knownin the art, such as those described in WO/2002/042480 and WO/2002/24224,the relevant disclosures of which are incorporated herein by references.

In another aspect, replication deficient MVA viral strains may also besuitable such as strain MVA-572, MVA-575 or any similarly attenuated MVAstrain. Also suitable can be a mutant MVA, such as the deletedchorioallantois vaccinia virus Ankara (dCVA). A dCVA comprises del I,del II, del III, del IV, del V, and del VI deletion sites of the MVAgenome. The sites are particularly useful for the insertion of multipleheterologous sequences. The dCVA can reproductively replicate (with anamplification ratio of greater than 10) in a human cell line (such ashuman 293, 143B, and MRC-5 cell lines), which then enable theoptimization by further mutation useful for a virus-based vaccinationstrategy (see WO 2011/092029).

In a preferred embodiment of the invention, the MVA vector(s) comprise anucleic acid that encode one or more antigenic proteins selected fromthe group consisting of GPs of Zaire ebolavirus (EBOV), GPs of Sudanebolavirus (SUDV), GPs of Marburg virus (MARV), the NP of Taï Forestvirus and GPs or NPs substantially similar thereto.

The filovirus protein can be inserted into one or more intergenicregions (IGR) of the MVA. In certain embodiments, the IGR is selectedfrom IGR07/08, IGR 44/45, IGR 64/65, IGR 88/89, IGR 136/137, and IGR148/149. In certain embodiments, less than 5, 4, 3, or 2 IGRs of therecombinant MVA comprise heterologous nucleotide sequences encodingantigenic determinants of a filovirus envelope glycoprotein and/or afurther filovirus protein. The heterologous nucleotide sequences may,additionally or alternatively, be inserted into one or more of thenaturally occurring deletion sites, in particular into the main deletionsites I, II, III, IV, V, or VI of the MVA genome. In certainembodiments, less than 5, 4, 3, or 2 of the naturally occurring deletionsites of the recombinant MVA comprise heterologous nucleotide sequencesencoding antigenic determinants of a filovirus envelope glycoproteinand/or a further filovirus protein.

The number of insertion sites of MVA comprising heterologous nucleotidesequences encoding antigenic determinants of a filovirus protein can be1, 2, 3, 4, 5, 6, 7, or more. In certain embodiments, the heterologousnucleotide sequences are inserted into 4, 3, 2, or fewer insertionsites. Preferably, two insertion sites are used. In certain embodiments,three insertion sites are used. Preferably, the recombinant MVAcomprises at least 2, 3, 4, 5, 6, or 7 genes inserted into 2 or 3insertion sites.

The recombinant MVA viruses provided herein can be generated by routinemethods known in the art. Methods to obtain recombinant poxviruses or toinsert exogenous coding sequences into a poxviral genome are well knownto the person skilled in the art. For example, methods for standardmolecular biology techniques such as cloning of DNA, DNA and RNAisolation, Western blot analysis, RT-PCR and PCR amplificationtechniques are described in Molecular Cloning, A laboratory Manual (2ndEd.) [J. Sambrook et al., Cold Spring Harbor Laboratory Press (1989)],and techniques for the handling and manipulation of viruses aredescribed in Virology Methods Manual [B. W. J. Mahy et al. (eds.),Academic Press (1996)]. Similarly, techniques and know-how for thehandling, manipulation and genetic engineering of MVA are described inMolecular Virology: A Practical Approach [A. J. Davison & R. M. Elliott(Eds.), The Practical Approach Series, IRL Press at Oxford UniversityPress, Oxford, UK (1993) (see, e.g., Chapter 9: Expression of genes byVaccinia virus vectors)] and Current Protocols in Molecular Biology[John Wiley & Son, Inc. (1998) (see, e.g., Chapter 16, Section IV:Expression of proteins in mammalian cells using vaccinia viral vector)].

For the generation of the various recombinant MVAs disclosed herein,different methods can be applicable. The DNA sequence to be insertedinto the virus can be placed into an E. coli plasmid construct intowhich DNA homologous to a section of DNA of the MVA has been inserted.Separately, the DNA sequence to be inserted can be ligated to apromoter. The promoter-gene linkage can be positioned in the plasmidconstruct so that the promoter-gene linkage is flanked on both ends byDNA homologous to a DNA sequence flanking a region of MVA DNA containinga non-essential locus. The resulting plasmid construct can be amplifiedby propagation within E. coli bacteria and isolated. The isolatedplasmid containing the DNA gene sequence to be inserted can betransfected into a cell culture, e.g., of chicken embryo fibroblasts(CEFs), at the same time the culture is infected with MVA. Recombinationbetween homologous MVA DNA in the plasmid and the viral genome,respectively, can generate an MVA modified by the presence of foreignDNA sequences.

According to a preferred embodiment, a cell of a suitable cell cultureas, e.g., CEF cells, can be infected with a poxvirus. The infected cellcan be, subsequently, transfected with a first plasmid vector comprisinga foreign or heterologous gene or genes, preferably under thetranscriptional control of a poxvirus expression control element. Asexplained above, the plasmid vector also comprises sequences capable ofdirecting the insertion of the exogenous sequence into a selected partof the poxviral genome. Optionally, the plasmid vector also contains acassette comprising a marker and/or selection gene operably linked to apoxviral promoter.

Suitable marker or selection genes are, e.g., the genes encoding thegreen fluorescent protein, β-galactosidase,neomycin-phosphoribosyltransferase or other markers. The use ofselection or marker cassettes simplifies the identification andisolation of the generated recombinant poxvirus. However, a recombinantpoxvirus can also be identified by PCR technology. Subsequently, afurther cell can be infected with the recombinant poxvirus obtained asdescribed above and transfected with a second vector comprising a secondforeign or heterologous gene or genes. In case, this gene shall beintroduced into a different insertion site of the poxviral genome, thesecond vector also differs in the poxvirus-homologous sequencesdirecting the integration of the second foreign gene or genes into thegenome of the poxvirus. After homologous recombination has occurred, therecombinant virus comprising two or more foreign or heterologous genescan be isolated. For introducing additional foreign genes into therecombinant virus, the steps of infection and transfection can berepealed by using the recombinant virus isolated in previous steps forinfection and by using a further vector comprising a further foreigngene or genes for transfection.

Alternatively, the steps of infection and transfection as describedabove are interchangeable, i.e., a suitable cell can at first betransfected by the plasmid vector comprising the foreign gene and, then,infected with the poxvirus. As a further alternative, it is alsopossible to introduce each foreign gene into different viruses,co-infect a cell with all the obtained recombinant viruses and screenfor a recombinant including all foreign genes. A third alternative isligation of DNA genome and foreign sequences in vitro and reconstitutionof the recombined vaccinia virus DNA genome using a helper virus. Afourth alternative is homologous recombination in E. coli or anotherbacterial species between a vaccinia virus genome, such as MVA, clonedas a bacterial artificial chromosome (BAC) and a linear foreign sequenceflanked with DNA sequences homologous to sequences flanking the desiredsite of integration in the vaccinia virus genome.

The heterologous filovirus gene can be under the control of (i.e.,operably linked to) one or more poxvirus promoters. In certainembodiments, the poxvirus promoter is a Pr7.5 promoter, a hybridearly/late promoter, or a PrS promoter, a PrS5E promoter, a synthetic ornatural early or late promoter, or a cowpox virus ATI promoter.

Immunogenic Compositions

Immunogenic compositions are compositions comprising an immunologicallyeffective amount of purified or partially purified adenovirus or MVAvectors for use in the invention. Said compositions can be formulated asa vaccine (also referred to as an “immunogenic composition”) accordingto methods well known in the art. Such compositions may includeadjuvants to enhance immune responses. The optimal ratios of eachcomponent in the formulation can be determined by techniques well knownto those skilled in the art in view of the present disclosure.

The preparation and use of immunogenic compositions are well known tothose of skill in the art. Liquid pharmaceutical compositions generallyinclude a liquid carrier such as water, petroleum, animal or vegetableoils, mineral oil or synthetic oil. Physiological saline solution,dextrose or other saccharide solution or glycols such as ethyleneglycol, propylene glycol or polyethylene glycol can be included.

The compositions of the invention may comprise any antigens. Theseantigenic peptides or polypeptides can include any sequences that areimmunogenic such as, for example, tumor-specific antigens (TSAs),bacterial, viral, fungal, and protozoal antigens.

The compositions of the invention may comprise filovirus antigens or thepriming or boosting inoculations may comprise other antigens. The otherantigens used in combination with the adenovirus vectors of theinvention are not critical to the invention and can be, for example,filovirus antigens and nucleic acids expressing them.

The immunogenic compositions useful in the invention can compriseadjuvants.

Adjuvants suitable for co-administration in accordance with theinvention should be ones that are potentially safe, well tolerated andeffective in people including QS-21, Detox-PC, MPL-SE, MoGM-CSF,TiterMax-G, CRL-1005, GERBU, TERamide, PSC97B, Adjumer, PG-026, GSK-I,GcMAF, B-alethine, MPC-026, Adjuvax, CpG ODN, Betafectin, Alum, andMF59.

Other adjuvants that can be administered include lectins, growthfactors, cytokines and lymphokines such as alpha-interferon, gammainterferon, platelet derived growth factor (PDGF), granulocyte-colonystimulating factor (gCSF), granulocyte macrophage colony stimulatingfactor (gMCSF), tumor necrosis factor (TNF), epidermal growth factor(EGF), IL-I, IL-2, IL-4, IL-6, IL-8, IL-IO, and IL-12 or encodingnucleic acids therefore.

The compositions of the invention can comprise a pharmaceuticallyacceptable excipient, carrier, buffer, stabilizer or other materialswell known to those skilled in the art. Such materials should benon-toxic and should not interfere with the efficacy of the activeingredient. The precise nature of the carrier or other material maydepend on the route of administration, e.g., intramuscular,subcutaneous, oral, intravenous, cutaneous, intramucosal (e.g., gut),intranasal or intraperitoneal routes.

Method for Enhancing an Immune Response

The invention provides an improved method of priming and boosting animmune response to any antigenic protein or immunogenic polypeptidethereof in a human subject using an MVA vector in combination with anadenoviral vector.

According to one general aspect of the invention, a method of enhancingan immune response in a human subject comprises:

-   -   a. administering to the human subject a first composition        comprising an immunologically effective amount of a MVA vector        comprising a first polynucleotide encoding an antigenic protein        or an immunogenic polypeptide thereof for priming the immune        response; and    -   b. administering to the subject a second composition comprising        an immunologically effective amount of an adenovirus vector        comprising a second polynucleotide encoding the antigenic        protein or an immunogenic polypeptide thereof for boosting the        immune response;    -   to thereby obtain an enhanced immune response against the        antigenic protein in the human subject.

According to embodiments of the invention, the enhanced immune responsecomprises an enhanced antibody response against the antigenic protein inthe human subject.

Preferably, the enhanced immune response further comprises an enhancedCD4+ response or an enhanced CD8+ T cell response against the antigenicprotein in the human subject. The enhanced CD4+ T cell responsegenerated by a method according to an embodiment of the invention canbe, for example, an increase or induction of a dominant CD4+ T cellresponse against the antigenic protein, and/or an increase or inductionof polyfunctional CD4+ T cells specific to the antigenic protein in thehuman subject. The polyfunctional CD4+ T cells express more than onecytokine, such as two or more of IFN-gamma, IL-2 and TNF-alpha. Theenhanced CD8+ T cell response generated by a method according to anembodiment of the invention can be, for example, an increase orinduction of polyfunctional CD8+ T cells specific to the antigenicprotein in the human subject.

More preferably, the enhanced immune response resulting from a methodaccording to an embodiment of the invention comprises an enhanced CD4+ Tcell response, an enhanced antibody response and an enhanced CD8+ T cellresponse, against the antigenic protein in the human subject.

In one or more embodiments of the invention, one or more MVA vectors areused to prime the immune response, and one or more rAd26 or rAd35vectors are used to boost the immune response.

The antigens in the respective priming and boosting compositions(however many boosting compositions are employed) need not be identical,but should share antigenic determinants or be substantially similar toeach other.

Administration of the immunogenic compositions comprising the vectors istypically intramuscular or subcutaneous. However other modes ofadministration such as intravenous, cutaneous, intradermal or nasal canbe envisaged as well. Intramuscular administration of the immunogeniccompositions can be achieved by using a needle to inject a suspension ofthe adenovirus vector. An alternative is the use of a needlelessinjection device to administer the composition (using, e.g.,Biojector(TM)) or a freeze-dried powder containing the vaccine.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the vector will be in the form of a parenterallyacceptable aqueous solution which is pyrogen-free and has suitable pH,isotonicity and stability. Those of skill in the art are well able toprepare suitable solutions using, for example, isotonic vehicles such asSodium Chloride Injection, Ringer's Injection, Lactated Ringer'sInjection. Preservatives, stabilizers, buffers, antioxidants and/orother additives can be included, as required. A slow-release formulationmay also be employed.

Typically, administration will have a prophylactic aim to generate animmune response against an antigen before infection or development ofsymptoms. Diseases and disorders that can be treated or prevented inaccordance with the invention include those in which an immune responsecan play a protective or therapeutic role. In other embodiments, the MVAand adenovirus vectors can be administered for post-exposureprophylactics.

The immunogenic compositions containing the MVA vectors are administeredto a subject, giving rise to an immune response in the subject. Anamount of a composition sufficient to in induce a detectable immuneresponse is defined to be an “immunologically effective dose.” As shownbelow, the immunogenic compositions of the invention induce a humoral aswell as a cell-mediated immune response. In a typical embodiment theimmune response is a protective immune response.

The actual amount administered, and rate and time-course ofadministration, will depend on the nature and severity of what is beingtreated. Prescription of treatment, e.g., decisions on dosage etc., iswithin the responsibility of general practitioners and other medicaldoctors, and typically takes account of the disorder to be treated, thecondition of the individual patient, the site of delivery, the method ofadministration and other factors known to practitioners. Examples of thetechniques and protocols mentioned above can be found in Remington'sPharmaceutical Sciences, 16th edition, Osol, A. ed., 1980.

Following production of MVA and adenovirus vectors and optionalformulation of such particles into compositions, the vectors can beadministered to an individual, particularly a human.

In one exemplary regimen, the adenovirus vector is administered (e.g.,intramuscularly) in a volume ranging between about 100 μl to about 10 mlcontaining concentrations of about 10⁴ to 10¹² virus particles/ml.Preferably, the adenovirus vector is administered in a volume rangingbetween 0.25 and 1.0 ml. More preferably the adenovirus vector isadministered in a volume of 0.5 ml.

Typically, the adenovirus is administered in an amount of about 10⁹ toabout 10¹² viral particles (vp) to a human subject during oneadministration, more typically in an amount of about 10¹⁰ to about 10¹²vp. In a preferred embodiment, the adenovirus vector is administered inan amount of about 5×10¹⁰ vp. In another preferred embodiment, theadenovirus vector is administered in an amount of about 0.8×10¹⁰ vp. Inanother preferred embodiment, the adenovirus vector is administered inan amount of about 2×10¹⁰ vp. In another preferred embodiment, theadenovirus vector is administered in an amount of about 4×10¹⁰ vp. Incertain embodiments, adenoviruses are formulated as a trivalentcomposition, wherein three adenoviruses with each a different insert,are mixed together. In a trivalent composition, each distinct adenovirusis preferably present in an amount of about 4×10¹⁰ vp. In said trivalentcomposition, the total number of adenovirus particles per dose amountsto about 1.2×10¹¹ vp. In another preferred embodiment, each distinctadenovirus in the trivalent composition is present in an amount of about1×10¹¹ vp. In said trivalent composition the total number of adenovirusparticles per dose then amounts to about 3×10¹¹ vp. The initialvaccination is followed by a boost as described above.

In one exemplary regimen, the MVA vector is administered (e.g.,intramuscularly) in a volume ranging between about 100 μl to about 10 mlof saline solution containing a dose of about 1×10⁷ TCID₅₀ to 1×10⁹TCID₅₀ (50% Tissue Culture Infective Dose) or Inf.U. (Infectious Unit).Preferably, the MVA vector is administered in a volume ranging between0.25 and 1.0 ml. More preferably the MVA vector is administered in avolume of 0.5 ml.

Typically, the MVA vector is administered in a dose of about 1×10⁷TCID₅₀ to 1×10⁹ TCID₅₀ (or Inf.U.) to a human subject during oneadministration. In a preferred embodiment, the MVA vector isadministered in an amount of about 5×10⁷ TCID₅₀ to 5×10⁸ TCID₅₀ (orInf.U.). In a more preferred embodiment, the MVA vector is administeredin an amount of about 5×10⁷ TCID₅₀ (or Inf.U.). In a more preferredembodiment, the MVA vector is administered in an amount of about 1×10⁸TCID₅₀ (or Inf.U.). In another preferred embodiment, the MVA vector isadministered in an amount of about 1.9×10⁸ TCID₅₀ (or Inf.U). In yetanother preferred embodiment, the MVA vector is administered in anamount of about 4.4×10⁸ TCID₅₀ (or Inf.U.). In a more preferredembodiment, the MVA vector is administered in an amount of about 5×10⁸TCID₅₀ (or Inf.U.)

The composition can, if desired, be presented in a kit, pack ordispenser, which can contain one or more unit dosage forms containingthe active ingredient. The kit, for example, may comprise metal orplastic foil, such as a blister pack. The kit, pack, or dispenser can beaccompanied by instructions for administration.

The compositions of the invention can be administered alone or incombination with other treatments, either simultaneously or sequentiallydependent upon the condition to be treated.

Boosting compositions are generally administered once or multiple times,weeks or months after administration of the priming composition, forexample, about 1 or 2 weeks or 3 weeks, or 4 weeks, or 6 weeks, or 8weeks, or 12 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or 28 weeks,or 32 weeks or one to two years.

Preferably, the initial boosting inoculation is administered 1-12 weeksor 2-12 weeks after priming, more preferably 1, 2, 4 or 8 weeks afterpriming. In a preferred embodiment, the initial boosting inoculation isadministered 4 or 8 weeks after priming. In additional preferredembodiments, the initial boosting is conducted at least 1 week, or atleast 2 weeks, or at least 4 weeks after priming. In still anotherpreferred embodiment, the initial boosting is conducted 4-12 weeks or4-8 weeks after priming.

In a more preferred embodiment according to this method, an MVA vectoris used for the priming followed by a boosting with an rAd26 vector.Preferably, the boosting composition is administered 1-12 weeks afterpriming, more preferably 1, 2, 4 or 8 weeks after priming. In apreferred embodiment, the boosting composition is administered 8 weeksafter priming. In another preferred embodiment, the boosting compositionis administered 1 week after priming. In another preferred embodiment,the boosting composition is administered 2 weeks after priming. Inanother preferred embodiment, the boosting composition is administered 4weeks after priming.

One or more additional boosting administrations can be performed afterthe initial boosting.

In a preferred embodiment, the boosting composition comprises an Ad26vector.

In one embodiment, the invention relates to a method of enhancing animmune response against a tumor in a human subject. The methodcomprises:

-   -   a. administering to the human subject a first composition        comprising an immunologically effective amount of a MVA vector        comprising a first polynucleotide encoding an antigenic protein        produced by a cell of the tumor, a substantially similar        antigenic protein, or an immunogenic polypeptide thereof for        priming the immune response; and    -   b. administering to the subject a second composition comprising        an immunologically effective amount of an adenovirus vector        comprising a second polynucleotide encoding the antigenic        protein, the substantially similar antigenic protein, or an        immunogenic polypeptide thereof for boosting the immune        response;    -   to thereby obtain an enhanced immune response against the tumor        in the human subject.

Preferably, the enhanced immune response provides the human subject witha protective immunity against the tumor.

In a preferred embodiment the boosting step is conducted 1-12 weeks or2-12 weeks after the first priming step. The boosting step can also beconducted later than 12 weeks after the priming step. In additionalpreferred embodiments, the boosting step is conducted at least 2 weeksor at least 4 weeks after the priming step. In still other preferredembodiments, the boosting step is conducted 4-12 weeks or 4-8 weeksafter the priming step.

In another embodiment, the boosting step is repeated one or more timesafter the initial boosting administration.

In another preferred embodiment, the adenovirus vector is an Ad26vector.

The antigenic protein produced by a cell of the tumor can be any tumorantigen. In a preferred embodiment, the tumor antigen is atumor-specific antigen that is present only on tumor cells. The tumorantigen can also be a tumor-associated antigen that is present on sometumor cells and also some normal cells.

According to another embodiment, the invention relates to a method ofenhancing an immune response against at least one subtype of filovirusin a human subject. The method comprises:

-   -   a. administering to the human subject a first composition        comprising an immunologically effective amount of a MVA vector        comprising a polynucleotide encoding an antigenic protein of the        at least one filovirus subtype, a substantially similar        antigenic protein, or an immunogenic polypeptide thereof, for        priming the immune response; and    -   b. administering to the subject a second composition comprising        an immunologically effective amount of an adenovirus vector        comprising a polynucleotide encoding an antigenic protein of the        at least one filovirus subtype, a substantially similar        antigenic protein, or an immunogenic polypeptide thereof, for        boosting the immune response;    -   to thereby obtain an enhanced immune response against the at        least one subtype of filovirus in the human subject.

Preferably, the enhanced immune response provides the human subject aprotective immunity against the at least one subtype of filovirus.

In a preferred embodiment the boosting step is conducted 1-12 weeks or2-12 weeks after the first step, more preferably 1, 2, 4, or 8 weeksafter priming. In additional preferred embodiments, the boosting step isconducted at least 1 week or at least 2 weeks after the priming. Instill other preferred embodiments, the boosting step is conducted 4-12weeks or 4-8 weeks after the priming.

The boosting step can also be conducted later than 12 weeks afterpriming.

In another embodiment, the boosting step is repeated one or more timesafter the initial boosting administration, such as 6 months, 1 year, 1.5years, 2 years, 2.5 years, or 3 years after priming.

In another preferred embodiment, the adenovirus vector is an Ad26vector.

In yet another preferred embodiment, the antigenic protein is aglycoprotein or a nucleoprotein of a filovirus subtype.

In one embodiment of the invention, the MVA vector in the firstcomposition comprises a polynucleotide encoding antigenic proteinsderived from more than one filovirus subtypes. More preferably, the MVAvector in the first composition comprises a polynucleotide encoding fourantigenic proteins from four filovirus subtypes having the amino acidsequences of SEQ ID NOs: 1, 2, 4 and 5, or immunogenic polypeptidesthereof.

In another embodiment of the invention, the second composition comprisesat least one adenovirus vector comprising a polynucleotide encoding anantigenic protein derived from a filovirus subtype that is same ordifferent from the filovirus subtype encoded by the MVA vector. Forexample, the adenovirus vector can comprise a polynucleotide encoding anantigenic protein having the amino acid sequence selected from the groupconsisting of SEQ ID NOs: 1-5. Preferably, the second composition cancomprise more than one adenovirus vectors encoding more than oneantigenic proteins or immunogenic polypeptides thereof from more thanone filovirus subtypes. For example, the second composition can compriseone to three adenovirus vectors encoding one to three of the antigenicproteins have the amino acid sequences of SEQ ID NOs: 1, 2 and 3.

EXAMPLES

The following examples are offered to illustrate, but not to limit theclaimed invention.

Example 1

An animal study was conducted with a goal of identifying a multivalentfilovirus vaccine with real efficacy 80% against multiple [e.g.,Marburg, Ebola (aka Zaire) & Sudan] Filoviruses for continued advancedevelopment. The study tested an extended vaccination schedule using twoor three vaccinations and impact of using heterologous (as opposed tohomologous) vaccine combinations on subsequent NHP immune responses tothe target Filoviruses. The vaccinated NHP was challenged with Ebolavirus Kikwit to test the efficacy of the applied vaccinations.

Animal Manipulations

These studies complied with all applicable sections of the Final Rulesof the Animal Welfare Act regulations (9 CFR Parts 1, 2, and 3) andGuide for the Care and Use of Laboratory Animals—National Academy Press,Washington D.C. Eight Edition (the Guide).

A total of 16 Cynomolgus macaques (Macaca fascicularis) (NHPs) (12 malesand 4 females), Mauritian-origin, cynomolgus macaques, 4-5 years old,approx. 4-8 kg each, were purchased from PrimGen (Hines, Ill.). Animalswere experimentally naive to Reston virus (RESTV) by ELISA prior tovaccination. Animals with prior exposure to Mycobacterium tuberculosis,Simian Immunodeficiency Virus (SIV), Simian T-Lymphotropic Virus-1(STLV-1), Macacine herpesvirus 1 (Herpes B virus), and Simian Retrovirus(SRV1 and SRV2) were excluded, and active infections with Salmonella andShigella were tested, and confirmed negative for Mycobacteriumtuberculosis.

Filoviruses are Risk Group 4 (High Containment) Pathogens; therefore allmanipulations involving Zaire ebolavirus, Sudan ebolavirus, orMarburgviruses were carried out in the CDC-accredited Biosafety Level(BSL)-4/Animal Biosafety Level (ABSL-4) containment facility.

Vaccine Materials

The rAd vectors were manufactured by Crucell Holland B.V. They arepurified E1/E3-deleted replication deficient recombinant Adenovirus type26 or type 35 vaccine vectors (Ad26 and Ad35, respectively) containingthe Filovirus Glycoprotein (GP) genes inserted at the E1 position. Thesevectors were rescued in PER.C6® cells, plaque purified, upscaled andthen purified by a two-step CsCl banding procedure and subsequentlyformulated in a TRIS-based formulation buffer and stored below −65° C.Release for in vivo use of these vectors includes bioburden test, lowendotoxin level (<5EU/ml) and confirmation of expression and integrityof the transgene.

In particular, the rAd vectors expressed EBOV Mayinga GP (SEQ ID NO:1),SUDV Gulu GP (SEQ ID NO:2) and MARV Angola GP (SEQ ID NO:3). Each rAdvector expressed one single antigenic protein (GP).

The MVA vectors were manufactured by Bavarian Nordic. In particular, theMVA-multi vector (MVA-BN-Filo) expressed 4 different antigenic proteins:EBOV Mayinga GP (SEQ ID NO:1); SUDV Gulu GP (SEQ ID NO:2); MARV MusokeGP (SEQ ID NO:4); and Taï forest virus (TAFV) NP (SEQ ID NO:5).

The vaccine materials were stored at −80° C. in a controlled temperaturefreezer.

Vaccination and Experimental Design

See FIGS. 1 and 2 for the study grouping and experimental design.

Cynomolgus macaques (Macaca fascicularis) (NHPs) were vaccinated usingtwo different vaccine platforms, 2 animals per group, in addition to acontrol group consisting of two naive (empty vector) challenge controls.Animals were first vaccinated with the recombinant vector(s) in groupsshown in FIG. 1. Each macaque was anesthetized and received anintramuscular (IM) injection of the vaccine into the left hind thigh.Priming and boosting doses were given 4 or 8 weeks apart (FIG. 1). Eachdose of adenoviruses consisted of a single IM injection into the leftposterior thigh. The MVA vectors were administered subcutaneously.

EDTA or heparin whole blood were shipped overnight at room temperatureto Texas Biomed on D28, D56 and D63. Additionally, Heparin or EDTA wholeblood was collected on D77 while animals were housed at Texas Biomed. Atall these time-points, EDTA whole blood will be processed for PBMC andplasma at Texas Biomed.

PBMC were used in an IFN-g ELISPOT assay using Ebola Zaire peptide pools1 and 2, Sudan Gulu peptide pools 1 and 2, an Ebolavirus consensuspeptide pool, Marburg Angola peptide pool 1 and 2 and a Marburgvirusconsensus peptide pool, together with a DMSO only negative control andan anti-CD3 stimulation positive control. All stimulations wereperformed in duplicate, for a total of 20 wells per NHP.

Additionally, whole blood without anticoagulant was processed for serumat Bioqual on D0, D28, D56 and D68, and on D77 at Texas Biomed. Aliquotsof the serum collected at Bioqual will be sent frozen to Texas Biomed onD68. Each serum was assayed in a ZEBOV GP specific ELISA. Additionally,serum from D0, D56 and D77 were assayed in a SEBOV GP and a MARVA GPspecific ELISA (two different assays).

TABLE 1 Parameters measured before challenge with Ebola virus Studyweeks Parameter 0 4 8 9 10 11 PBMC and plasma processing X X X X ZEBOVGP ELISA—all animals X X X X X SEBOV GP ELISA—all animals X X X MARVA GPELISA—all animals X X X Filovirus ELISPOT—all animals X X X X

Filovirus Inoculum for Animal Challenges

As shown in FIG. 2, about 4 weeks after the boosting vaccination, theanimals were challenged with EBOV. In particular, EBOV kikwit-9510621was used for animal challenges and was supplied by Texas Biomed. Asecond cell-culture passage (P2) of EBOV kikwit-9510621 was obtainedfrom Dr. Tom Ksiazek (at NIAID's WRCEVA at UTMB's Health GalvestonNational Laboratory) in 2012 and propagated at Texas Biomed for a thirdtime in Vero E6 cells and had a titer of 2.1×10⁵ PFU/ml. EBOVkikwit-9510621. LOT No. 2012099171.

Titer at harvest: 2.1×10⁵ PFU/ml was used for the study.

The challenge stock has been confirmed to be wild-type EBOV kikwit9510621 by deep sequencing with only 1 SNP difference from the GenbankP2 consensus sequence. The challenge stock was stored in liquid nitrogenvapor phase as 500±50 μl aliquots containing media (MEM) containing 10%FBS. For a 100 PFU challenge, the filovirus challenge agent was dilutedto a target dose of 200 PFU/ml in phosphate buffered saline. Briefly,stock virus was diluted via three consecutive 1:10 dilutions in PBS toachieve a 200 PFU/ml challenge material concentration. A total of 0.5 mlof challenge material was given to each animal.

Prior to virus injection, monkeys were sedated via intramuscularinjection with Telazol (2 to 6 mg/kg; 5 to 8 mg/kg ketamine IM prn forsupplemental anesthesia). On Study Day 0, blood was collected and eachmonkey was subsequently challenged with a targeted dose of 100 PFU ofEBOV in a 0.5 ml volume via intramuscular injection in the right deltoidmuscle of the arm. The challenge site was recorded.

Following virus administration, each monkey was returned to its homecage and observed until it has recovered from anesthesia (sternalrecumbancy/ability to maintain an upright posture). Endpoints in thisstudy were survival/nonsurvival. Nonsurvival is defined by an animalhaving terminal illness or being moribund. Animals' health was evaluatedon a daily clinical observation score sheet.

Anti-EBOV GP IgG ELISA

Filovirus-specific humoral response was determined at time pointsdescribed in table 1 by a modified enzyme-linked immunosorbent assay(ELISA), as previously described in Sulivan et al. (2006) (Immuneprotection of nonhuman primates against Ebola virus with single low-doseadenovirus vectors encoding modified GPs. PLoS Medicine 3, e177), whichis incorporated by reference herein in its entirety. Briefly, ELISAplates were coated over night with Galanthus Nivalis Lectin at 10 μg/ml.Then, after blocking, the plates were coated with either an Ebola or aMarburg strain specific GP supernatant. These supernatants were producedby transient transfection of Hek293T with expression plasmids coding forfilovirus glycoprotein deleted of the transmembrane domain andcytoplasmic tail. Monkey serum samples were tested in a 4-fold dilutionseries starting at 1:50. Bound IgG was detected by colorimetry at 492nm. Relative serum titers were calculated against a filovirusglycoprotein strain specific reference serum. The results of the Elisaassay are shown in FIGS. 4-6.

IFN-g ELISPOT Assay

Filovirus-specific cellular immune response was determined at timepoints described in table 1 by interferon gamma Enzyme-linked immunospotassay (ELISPOT) as previously described in Ophorst et al. 2007(Increased immunogenicity of recombinant Ad35-based malaria vaccinethrough formulation with aluminium phosphate adjuvant. Vaccine 25,6501-6510), which is incorporated by reference herein in its entirety.The peptide pools used for stimulation for each Ebola and Marburg strainglycoprotein consist of 15-mers overlapping by 11 amino acids. Tominimize undesired effects of a too high number of peptides in a pool,each glycoprotein peptide pool was divided into two, one N-terminal andone C-terminal half.

Peptides that overlap with more than nine consecutive amino acids withinthree Ebolavirus (Zaire, Sudan and Tat Forest) or two Marburgvirus(Marburg and Ravn viruses) were combined in a consensus pool. Thepeptide pools and single peptides were used at a final concentration of1 μg/ml for each single peptide. The results of the ELISPOT assay areshown in FIG. 7.

As shown by results summarized in FIGS. 3-7, the animal study hereindemonstrated the utility of rAd and MVA vectors in prime-boostcombinations for preventing filovirus infections in primates. Inparticular, the administration of one or more rAd26 vectors expressingGP(s) of one or more types of filoviruses or MVA vectors expressingmultiple filovirus antigens resulted in efficient priming of the humoralresponse to the one or more types of filoviruses. After boostimmunization at week 8 with the heterologous vector, all vaccine regimesinduced a similar humoral and cellular immune response to the one ormore types of filoviruses and provided 100% protection against a highlypathogenic Ebola Zaire challenge.

Example 2

A second NHP study was performed to confirm the immunogenicity andprotective efficacy of 2 prime-boost regimens at 0-4 week and at 0-8week intervals. One comprising a monovalent Ad26.ZEBOV vaccine as aprime and a MVA-BN-Filo as a boost; the other one comprising aMVA-BN-Filo as a prime and an Ad26.ZEBOV as a boost. All immunizationswere Intra muscular. Ad26.ZEBOV (5×10¹⁰ vp) was used as a prime for the0-8 week regimen, and was combined with a boost of 1×10⁸ TCID₅₀ ofMVA-BN-Filo (4 NHPs) and 5×10⁸ TCID₅₀ MVA-BN-Filo (4 NHPs) to assess theimpact of a standard and a high dose of MVA in this regimen. Twoadditional groups of 4 NHPs were primed with 1×10⁸ TCID₅₀ of MVA-BN-Filoand 5×10⁸ TCID₅₀ MVA-BN-Filo, respectively; in both cases followed by aboost with Ad26.ZEBOV (5×10¹⁰ vp) after 4 weeks, to test the impact ofthe MVA-BN-Filo dose as a prime in a 4-week regimen. In addition, 2 NHPswere primed with Ad26.ZEBOV (5×10¹⁰ vp) followed by 1×10⁸ TCID₅₀ ofMVA-BN-Filo. Finally, 2 NHPs were immunized with empty Ad26 vector (notexpressing any Filovirus antigens, 5×10¹⁰ vp IM) and TBS as negativeimmunization control for the study. All animals were challenged 4 weeksafter the last immunization with 100 pfu of EBOV Kikwit 1995 wild-typeP3 challenge virus. The grouping of this study is summarized in Table 2.

TABLE 2 Experimental Grouping of Protection Study in Non-human PrimatesChallenged With EBOV Immunization 1 Immunization 2 ImmunizationChallenge Survival Group (Dose 1) (Dose 2) Schedule (Weeks) After 4Weeks Ratio (%) 1/A Ad26.empty MVA negative 0-8 EBOV (Kikwit) 0/2 (0%)(5 × 10¹⁰ vp) control (TBS) 2/B Ad26.ZEBOV MVA-BN-Filo 0-8 EBOV (Kikwit)4/4 (100%) (5 × 10¹⁰ vp) (5 × 10⁸ TCID₅₀) 3/C MVA-BN-Filo Ad26.ZEBOV 4-8EBOV (Kikwit) 2/4 (50%) (5 × 10⁸ TCID₅₀) (5 × 10¹⁰ vp) 4/D Ad26.ZEBOVMVA-BN-Filo 0-8 EBOV (Kikwit) 4/4 (100%) (5 × 10¹⁰ vp) (1 × 10⁸ TCID₅₀)5/E MVA-BN-Filo Ad26.ZEBOV 4-8 EBOV (Kikwit) 2/4 (50%) (1 × 10⁸ TCID₅₀)(5 × 10¹⁰ vp) 6/F Ad26.ZEBOV MVA-BN-Filo 4-8 EBOV (Kikwit) 2/2 (100%) (5× 10¹⁰ vp) (1 × 10⁸ TCID₅₀) Abbreviations: TBS: Tris-buffered saline;TCID₅₀: 50% tissue culture infective dose; vp: viral particles. 100%survival are in bold.

Immunogenicity

The immune response in NHP is characterized with respect to FilovirusGP-binding and neutralizing antibodies (ELISA) as well as cytokineproducing T cells (ELISpot).

ELISA:

EBOV Mayinga GP reactive antibodies were analyzed by GP-specific ELISAfor all timepoints (see FIG. 20). The Anti-EBOV GP IgG ELISA wasperformed as described in experiment 1. ELISA titers were not observedin control-vaccinated animals. The vaccine regimens were immunogenic inall animals. The highest titers were observed in Group B, receivingAd26.ZEBOV and a high dose of MVA-BN-Filo with an 8-week interval.

Protective Efficacy

Both 8-week Ad26.ZEBOV/MVA-BN-Filo prime/boost regimens resulted incomplete survival after EBOV challenge, irrespective of the dose ofMVA-BN-Filo (1×10⁸ TCID₅₀ or 5×10⁸ TCID₅₀). Additionally, a 4-weekregimen of Ad26.ZEBOV/MVA-BN-Filo gave protection in 2 out of 2 NHPs.Both 4-week MVA-BN-Filo/Ad26.ZEBOV regimens gave protection in 2 out of4 NHPs.

Example 3

A clinical study is performed in humans for evaluating the safety,tolerability and immunogenicity of regimens using MVA-BN-Filo at a doseof 1×10⁸ TCID₅₀ and Ad26.ZEBOV at a dose of 5×10¹⁰ vp. The studyconsisted of two parts.

The main study is a randomized, placebo-controlled, observer-blind studybeing conducted in 72 healthy adult subjects who never received anexperimental Ebola candidate vaccine before and have no known exposureto an Ebola virus or diagnosis of Ebola disease. In this study 4regimens are tested: 2 regimens have MVA-BN-Filo as prime and Ad26.ZEBOVas boost at a 28- or 56-day interval, and 2 regimens have Ad26.ZEBOV asprime and MVA-BN-Filo as boost at a 28- or 56-day interval.

The sub-study consists of an open-label, uncontrolled non-randomizedtreatment arm evaluating the safety, tolerability and immunogenicity ofa regimen with Ad26.ZEBOV at a dose of 5×10¹⁰ vp as prime, andMVA-BN-Filo at a dose of 1×10⁸ TCID₅₀ as boost 14 days later, and isconducted in 15 healthy adult subjects.

The study consists of a vaccination period in which subjects arevaccinated at baseline (Day 1) followed by a boost on Day 15, 29 or 57,and a post-boost follow-up until all subjects have had their 21-daypost-boost visit (Day 36, 50 or 78) or discontinued earlier.

Subjects in the main study are enrolled into 4 different groups of 18healthy subjects each. Overall, subjects are randomized within a groupin a 5:1 ratio to receive active vaccine or placebo (0.9% saline)through IM injections (0.5 ml) as follows:

-   -   MVA-BN-Filo (1×10⁸ TCID₅₀) on Day 1, followed by a booster of        Ad26.ZEBOV (5×10¹⁰ vp) on Day 29 (Group 1) or Day 57 (Group 2),        or    -   Ad26.ZEBOV (5×10¹⁰ vp) on Day 1, followed by a booster of        MVA-BN-Filo (1×10⁸ TCID₅₀) on Day 29 (Group 3) or Day 57 (Group        4).        The 15 subjects in the substudy receive active vaccine through        IM injections (0.5 ml) as follows:    -   Ad26.ZEBOV (5×10¹⁰ vp) on Day 1, followed by a booster of        MVA-BN-Filo (1×10⁸ TCID₅₀) on Day 15 (Group 5).        The exemplary study vaccination schedules are summarized in        Table 3.

TABLE 3 Study Vaccination Schedules Group N Day 1 Day 15 Day 29 Day 57 118 15 MVA-BN-Filo — Ad26.ZEBOV — 1 × 10⁸ TCID₅₀ 5 × 10¹⁰ vp 3 placebo(0.9% — placebo (0.9% — saline) saline) 2 18 15 MVA-BN-Filo — —Ad26.ZEBOV 1 × 10⁸ TCID₅₀ 5 × 10¹⁰ vp 3 placebo (0.9% — — placebo (0.9%saline) saline) 3 18 15 Ad26.ZEBOV — MVA-BN-Filo — 5 × 10¹⁰ vp 1 × 10⁸TCID₅₀ 3 placebo (0.9% — placebo (0.9% — saline) saline) 4 18 15Ad26.ZEBOV — — MVA-BN-Filo 5 × 10¹⁰ vp 1 × 10⁸ TCID₅₀ 3 placebo (0.9% —— placebo (0.9% saline) saline) 5 15 Ad26.ZEBOV MVA-BN-Filo — — 5 × 10¹⁰vp 1 × 10⁸ TCID₅₀ N: number of subjects to receive study vaccine;TCID₅₀: 50% Tissue Culture Infective Dose; vp: viral particles

Safety is assessed by collection of solicited local and systemic adverseevents, unsolicited adverse events and serious adverse events, and byphysical examination. In addition, standard chemistry, hematologic(including coagulation parameters) and urinalysis parameters areassessed at multiple time points.

Immunogenicity is assessed using the immunologic assays summarized inTables 4 and 5. The exploratory assay package may include, but is notlimited to, the listed assays.

TABLE 4 Summary of Immunologic Assays (Serology) Assay Purpose Secondaryendpoints Virus neutralization Analysis of neutralizing antibodies toEBOV GP assay ELISA Analysis of antibodies binding to EBOV GPExploratory endpoints Adenovirus/MVA Neutralizing antibodies toadenovirus/MVA neutralization assay Molecular antibody Analysis ofanti-EBOV GP, SUDV characterization GP, MARV GP and/or TAFV NP antibodycharacteristics, including IgG subtyping Exploratory ELISA Analysis ofbinding antibodies to a different source of EBOV GP EBOV: Ebola virus;ELISA: enzyme-linked immunosorbent assay; GP: glycoprotein; IgG:Immunoglobulin G; MARV: Marburg virus; MVA: Modified Vaccinia Ankara;NP: nucleoprotein; SUDV: Sudan virus; TAFV: TaÏ Forest virus

TABLE 5 Summary of Immunologic Assays (Cellular) Assay Purpose Secondaryendpoints ELISpot T-cell IFN-γ responses to EBOV GP Exploratoryendpoints ICS of frozen PBMC Analysis of T-cell responses to EBOV GP,SUDV GP, MARV GP and/or TAFV NP (including CD4/8, IL-2, IFN-γ, TNF-αand/or activation markers) ICS and/or ELISpot Analysis of T cellresponses to EBOV GP including CD4-positive and low- of fresh PBMCmagnitude T cell responses EBOV: Ebola virus; ELISpot: enzyme-linkedimmunospot; GP: glycoprotein; ICS: intracellular cytokine staining; IFN:interferon; IL: interleukin; MARV: Marburg virus; NP: nucleoprotein;PBMC: peripheral blood mononuclear cells; SUDV: Sudan virus; TAFV:Ta{umlaut over (l)} Forest virus; TNF: tumor necrosis factor

Safety Assessment

Safety was assessed by collection of solicited local and systemicadverse events, unsolicited adverse events and serious adverse events,and by physical examination. In addition, standard chemistry,hematologic (including coagulation parameters) and urinalysis parameterswere assessed at multiple time points.

The safety data from this first in human showed that both vaccinesappear to be well-tolerated at this stage with transient reactionsnormally expected from vaccination. No significant adverse events wereassociated with the vaccine regimen. The majority of events were mild,occurring one to two days post-vaccination, and lasting one to two dayson average. Very few cases of fever were observed.

Assessment of Immune Response

Immunogenicity was assessed up to 21 days post-boost immunization usingan ELISA assay to analyze antibodies binding to EBOV GP, an ELISpotassay to analyze an EBOV GP-specific T cell response, and ICS assays todetect CD4+ and CD8+ T-cell responses to EBOV GP. Samples for theanalysis of the humoral and cellular immune response induced by thestudy vaccines were collected on Days 1, 8, 29, 36 and 50 in Groups 1and 3 and on Days 1, 8, 29, 57, 64 and 78 for Groups 2 and 4.

Assessment of Humoral Immune Response

The binding antibody responses induced by study vaccines was assessed byan anti-EBOV GP ELISA assay (FIG. 8). Importantly, all subjects who havereceived a vaccine regimen showed seroconversion at 21 days post-boostimmunization. While the EBOV GP-specific immune response post-prime withMVA-BN-Filo was only observed at low levels in 7 to 40% of the subjects,a strong antigen-specific response was observed post-boost withAd26.ZEBOV administered at 28 days or 56 days post-prime. Surprisingly,this response is of higher magnitude than the one induced by the reversevaccine regimen at the same prime-boost time interval (Group 3 and Group4, Ad26.ZEBOV prime followed by MVA-BN-Filo boost 28 or 56 days later,respectively) at 21 days post-boost immunization [Geometric mean titerswith 95% confidence interval of EU/mL 10573 (6452; 17327) and 4274(2350; 7775) for groups 1 and 3, respectively, and 18729 (12200; 28751)and 7553 (511; 1115) for groups 2 and 4, respectively].

It must be noted that in nonhuman primate (NHP), boosting an MVA primewith Ad26 had resulted in an EBOV GP-specific immune response, that wascomparable in magnitude to that induced by the reverse vaccine regimen(Ad/MVA), at the same prime-boost time interval (see FIG. 4) or that wasinferior in magnitude (FIG. 20). Thus, the immune responses observed forone specific prime-boost regimen in NHP were not predictive for theimmune responses observed following that same prime-boost regimen inhumans.

Assessment of Cellular Immune Response

The EBOV GP-specific cellular immune response was measured by interferongamma (IFN-γ) ELISpot and ICS. To assess the cellular immune response,stored PBMC (peripheral blood mononuclear cells) were thawed andstimulated with peptides organized in 2 pools (Pools 1 and 2). The sumof the T-cell responses stimulated per pool are shown in FIG. 9.

By ELISpot analysis (FIG. 9), an IFN-γ response could readily bedetected in 50 to 60% of the subjects at day 29 after prime immunizationwith Ad26.ZEBOV (median IFN-γ response 103 and 58 spots forming unitsper million PBMC for Group 3 and 4, respectively) and in 86% of thesubjects at day 57 post Ad26.ZEBOV prime immunization (Group 4, medianIFN-γ response 283 spots forming units per million (SFU/10⁶) PBMC).These responses were further boosted by immunization on Day 29 or Day 57with MVA-BN-Filo (87% responders, median IFN-γ response 463 SFU/10⁶ PBMCfor Group 3, 86% responders, 648 SFU/10⁶ PBMC for Group 4) andmaintained at that level up to day 21 post-boost (79% responders, medianIFN-γ response 390 SFU/10⁶ PBMC for Group 3 and 100% responders, 464SFU/10⁶ PBMC for Group 4).

By contrast, only a very low level of EBOV GP-specific IFN-γ secretingcells could be detected post-MVA prime (7% and 0% responders at Day 29for Group 1 and 2, respectively). However, a strong IFN-γ response wasunexpectedly observed peaking at 7 days post-Ad26.ZEBOV boost in 93% and100% of the subjects boosted at day 29 and day 57, respectively (medianIFN-γ response 882 and 440 SFU/10⁶PBMC), at a level higher than thatobserved after the Ad26.ZEBOV-prime/MVA-BN-Filo-boost combination usingthe same time schedule (Group 3 and Group 4).

Results for the cellular assays measuring specific CD4+ and CD8+ T cellresponses by ICS are shown in FIGS. 10-15.

As expected, no EBOV GP-specific CD8+ or CD4+ T cell response wasobserved in placebo immunized individuals (FIGS. 10 and 13). No CD8+cytokine responses were observed on Day 29 or Day 57 after primeimmunization with MVA-BN-Filo (Group 1 and 2). However, avaccine-induced CD8+ T cell response was observed in 53% of subjects 7days post-boost with Ad26.ZEBOV (median total cytokine response: 0.08%and 0.07%, when Ad26 boost immunization administered at day 29 or day57, respectively; FIG. 10). This response was maintained at day 21 postboost immunization (median total cytokine response: 0.1% and 0.06%, whenAd26 boost immunization administered at day 29 or day 57, respectively;FIG. 10). In comparison, 57% of subjects receiving a prime immunizationwith Ad26.ZEBOV (Group 3 and Group 4) showed at Day 29 a CD8+ T cellresponse (median total cytokine response: 0.12 and 0.05%, respectively),86% of the subjects at day 57 post Ad26.ZEBOV prime immunization (Group4) showed a CD8+ T cell response (median total cytokine response:0.19%). This response was further enhanced after boost immunization withMVA-BN-Filo on day 29, with 67% and 73% of subjects responding 7 and 21days post-boost, respectively (median response: 0.27% on both days).

Surprisingly, while a smaller percentage of responders was observed inGroup 1 (MVA-Ad26 prime-boost 0-28 day schedule) compared to Group 3(Ad26-MVA prime-boost 0-28 day schedule), the proportion ofpolyfunctional CD8+ T cells (CD8+ T cells expressing more than onecytokine) induced by the MVA-Ad26 prime-boost regimen in theseresponders was higher post-boost than that induced by the Ad26-MVAprime-boost regimen (FIG. 11). This difference was not observed when theprime and boost were administered at day 57. Using this schedule, boththe MVA prime Ad26 boost (Group 2) and the Ad26 prime MVA boost (Group4) regimens induced similarly high proportion of polyfunctional CD8+ Tcells (FIG. 12).

Surprisingly, prime immunization with MVA-BN-Filo followed by a boostwith Ad26.ZEBOV given at 28 days interval (Group 1) induced a veryrobust CD4+ T cell response which peaked 7 days post-boost immunization(93% responders, median total cytokine response 0.37%; FIG. 13). At thepeak, this CD4+ T cell response was of a higher magnitude than that seenin Group 3 after prime immunization with Ad26.ZEBOV followed by aMVA-BN-Filo boost at 28 days interval (67% responders, median totalcytokine response 0.11%). 21 days post-boost, the CD4+ T cell responsesinduced by both regimens were comparable. Extending the interval of theMVA-BN-Filo/Ad26.ZEBOV regimen to 56 days resulted in lower CD4+ T cellresponses. The Ad26.ZEBOV/MVA-BN-Filo regimen induced slightly lowerCD4+ T cell responses at a 28-day interval and comparable responses at a56-day interval. The CD4+ T cells induced by both vaccine combinationswere predominantly polyfunctional (FIGS. 14 and 15).

Results of the substudy assessing the immunogenicity of a prime withAd26.ZEBOV at 5×10¹⁰ vp followed by a boost 14 days later using 1×10⁸TCID₅₀ of MVA-BN-Filo are summarized below.

Overall, this relatively short regimen using a 14-days interval betweenprime and boost has been shown to be immunogenic. The humoral immuneresponse to vaccinations was assessed by ELISA. As observed for longerintervals, all subjects seroconverted by 21 days post boost immunization(FIG. 16A). Furthermore, a cellular immune response was observed byELISpot in 92% of the subjects 21 days post boost immunization (FIG.16B). This cellular immune response consisted of both CD4+ (67%responders, median response 0.08% at day 21 post boost) and CD8+ (64%responders, median response 0.15% at day 7 post boost) specific T cells.The immune response induced using a 2 weeks interval appeared somewhatlower than the response induced when using longer intervals betweenprime and boost (refer to previous section).

Example 4

A randomized, placebo-controlled, observer-blind study (preceded by aninitial open-label vaccination of a total of 6 sentinel study subjects)is performed to evaluate the safety, tolerability and immunogenicity ofa heterologous regimen of (a) a single dose of MVA-BN-Filo (1×10⁸TCID₅₀) or placebo (0.9% saline) as prime followed by a single dose ofAd26.ZEBOV (5×10¹⁰ vp) or placebo as boost at different time points (14,28, or 56 days after prime; Groups 1 to 3) and (b) a single dose ofAd26.ZEBOV (5×10¹⁰ vp) or placebo as prime followed by a single dose ofMVA-BN-Filo (1×10⁸ TCID₅₀) or placebo as boost at 28 days after prime(Group 4).

In order to assess the safety of the 2 vaccines independently, Groups 5and 6 are included where homologous regimens of 2 single doses ofMVA-BN-Filo (1×10⁸ TCID₅₀) or placebo, or 2 single doses of Ad26.ZEBOV(5×10¹⁰ vp) or placebo are administered with the shorter prime-boostschedule of 1 and 15 days. This study is conducted in a target ofapproximately 92 healthy subjects, aged between 18 and 50 years(inclusive) who have never received an experimental Ebola candidatevaccine before and have no known exposure to or diagnosis of Eboladisease.

The study consists of a vaccination period in which subjects arevaccinated at their baseline visit (Day 1) followed by a boost on Day15, 29, or 57, and a post-boost follow-up period until all subjects havehad their 21-day post-boost visit, or discontinued earlier. At thattime, the study will be unblinded.

Subjects are enrolled in 6 different groups, comprising 18 (Groups 1 to4) or 10 (Groups 5 and 6) healthy subjects each. Within Groups 1 to 4,subjects are randomized in a 5:1 ratio to receive active vaccine orplacebo throughout the study. Groups 5 and 6 each start with a SentinelCohort of 3 subjects who receive active vaccine in an open-labelfashion, followed by a blinded cohort of 7 subjects, who are randomizedin a 6:1 ratio to receive active vaccine or placebo.

The study vaccination schedules in the different groups are summarizedin Table 6.

TABLE 6 Study Vaccination Schedules Group N n Day 1 Day 15 Day 29 Day 571 18 15 MVA-BN-Filo Ad26.ZEBOV 3 Placebo Placebo 2 18 15 MVA-BN-FiloAd26.ZEBOV 3 Placebo Placebo 3 18 15 MVA-BN-Filo Ad26.ZEBOV 3 PlaceboPlacebo 4 18 15 Ad26.ZEBOV MVA-BN-Filo 3 Placebo Placebo 5 10 3MVA-BN-Filo MVA-BN-Filo (sentinel) (sentinel) 6 MVA-BN-Filo MVA-BN-Filo1 Placebo Placebo 6 10 3 Ad26.ZEBOV Ad26.ZEBOV (sentinel) (sentinel) 6Ad26.ZEBOV Ad26.ZEBOV 1 Placebo Placebo N: number of subjects to receivestudy vaccine MVA-BN-Filo dose level is 1 × 10⁸ TCID₅₀ (50% TissueCulture Infective Dose) in all groups; Ad26.ZEBOV dose level is 5 × 10¹⁰vp (viral particles) in all groups; Placebo is 0.9% saline

Safety is assessed by collection of solicited local and systemic adverseevents, unsolicited adverse events and serious adverse events, and byphysical examination. In addition, standard chemistry, hematologic(including coagulation parameters) and urinalysis parameters areassessed at multiple time points.

Immunogenicity is assessed using the immunologic assays summarized inTable 7 and 8. The exploratory assay package may include, but is notlimited to, the listed assays.

TABLE 7 Summary of Immunologic Assays (Serology) Assay Purpose Secondaryendpoints Virus neutralization Analysis of neutralizing antibodies toEBOV GP assay ELISA Analysis of antibodies binding to EBOV GPExploratory endpoints Adenovirus/MVA Neutralizing antibodies toadenovirus/MVA neutralization assay Molecular antibody Analysis ofanti-EBOV GP, SUDV characterization GP, MARV GP and/or TAFV NP antibodycharacteristics, including IgG subtyping Exploratory ELISA Analysis ofbinding antibodies to a different source of EBOV GP EBOV: Ebola virus;ELISA: enzyme-linked immunosorbent assay; GP: glycoprotein; IgG:Immunoglobulin G; MARV: Marburg virus; MVA: Modified Vaccinia Ankara;NP: nucleoprotein; SUDV: Sudan virus; TAFV: TaÏ Forest virus

TABLE 8 Summary of Immunologic Assays (Cellular) Assay Purpose Secondaryendpoints ELISpot T-cell IFN-γ responses to EBOV GP Exploratoryendpoints ICS of frozen PBMC Analysis of T-cell responses to EBOV GP,SUDV GP, MARV GP and/or TAFV NP (including CD4/8, IL-2, IFN-γ, TNF-αand/or activation markers) ICS and/or ELISpot Analysis of T cellresponses to EBOV GP of fresh PBMC including CD4-positive and low-magnitude T cell responses EBOV: Ebola virus; ELISpot: enzyme-linkedimmunospot; GP: glycoprotein; ICS: intracellular cytokine staining; IFN:interferon; IL: interleukin; MARV: Marburg virus; NP: nucleoprotein;PBMC: peripheral blood mononuclear cells; SUDV: Sudan virus; TAFV: ÏForest virus; TNF: tumor necrosis factorThe clinical study is ongoing. Some of the initial results are describedbelow.

Assessment of Humoral Immune Response

As shown in FIG. 17, all subjects seroconverted 21 days post boostimmunization when assessed by ELISA. Similar to previous experiments, ahigher immune response was observed at 21 days post boost immunizationwhen MVA was used as a prime and Ad26 administered as a boost 28 dayslater (Group 2, Geometric Mean Concentration of EU/mL 6987) compared tothe reverse order of vaccine immunization (Group 4, Geometric MeanConcentration of EU/mL 2976).

The strength of the humoral immune response correlated with the intervalbetween the prime and the boost, with higher antibody concentrationsobserved when using a 56 days interval between MVA prime and Ad26 boost(group 3, Geometric Mean Concentration of EU/mL 14048) compared to ashorter schedule (group 1, 14 days interval, Geometric MeanConcentration of EU/mL 4418 and group 2, 28 days interval, GeometricMean Concentration of EU/mL 6987).

Surprisingly, a robust humoral immune response as assessed by ELISA wasobserved when MVA-BN-Filo was used as a prime and followed by a boostimmunization with Ad26.ZEBOV 14 days later. All subjects receiving thevaccine regimen seroconverted by 21 days post boost immunization, andthe antibody concentration at this time point reached similar or higherlevels than when using the Ad26 prime MVA boost combination at a 28 dayintervals (Geometric Mean Titer of EU/mL 4418 and 2976, respectively).Surprisingly, the antibody concentrations induced by this MVA/Ad26 primeboost combination at 14 days interval Were strikingly higher than theresponse induced by the reverse vaccine regimen at the same prime-boosttime interval (refer to example 2, FIG. 16A, Geometric MeanConcentration of EU/mL 915). This confirms the induction of a robustimmune response by an MVA prime Ad26 boost combination and the advantageof such combination when using a short prime boost interval (14 days).

Assessment of Cellular Immune Response

The EBOV GP-specific cellular immune response was measured by interferongamma (IFN-γ) ELISpot and ICS. To assess the cellular immune response,stored PBMC (peripheral blood mononuclear cells) were thawed andstimulated with peptides organized in 2 pools (Pools 1 and 2). The sumof the T cell responses stimulated per pool are shown in FIGS. 18-19.

Surprisingly, when using MVA-BN-Filo as a prime followed by Ad26.ZEBOVas a boost, a stronger IFN-γ response was observed when using a shorter14 days interval between prime and boost (87 and 93% responders, 395 and577 SFU/10⁶ PBMC for Group 1 at day 7 and 21 post boost, respectively)compared to the response induced by a 28 days (Group 2, 73 and 67%responders, median IFN-γ response 427 and 375 SFU/10⁶ PBMC for day 7 andday 21 post boost) or 56 days interval (Group 3, 47% responders, medianIFN-γ response 118 and 153 SFU/10⁶ PBMC for day 7 and day 21 postboost).

Remarkably, the cellular immune response induced by the MVA-BN-Filoprime Ad26.ZEBOV boost at a 14 days interval was well balanced with bothEBOV GP-specific CD8+ and CD4+ T cell response (73% responders for bothCD4+ and CD8+ T cells, CD4+ median total cytokine response 0.15 and0.19% at day 7 and 21 post boost, respectively; CD8+ median totalcytokine response 0.19 and 0.34% at day 7 and 21 post boost,respectively; FIG. 19 A and B). Both the CD8+ and CD4+ T cells inducedby this vaccine combination were predominantly polyfunctional (FIG. 19 Cand D).

Unexpectedly, the cellular immune response induced by this MVA/Ad26prime boost combination at 14 days interval were strikingly higher thanthe response induced by the reverse vaccine regimen using the sameprime-boost interval (refer to example 2, FIG. 16 B, C and D). Thisconfirms the potential of an MVA prime Ad26 boost combination when usinga short prime boost time interval (14 days).

The following tables 9-12 are presented as summaries of the clinicalstudies presented herein. The studies presented in example 3 and 4 arenumbered study 1001 and 1002 respectively.

Table 9 is a summary of the humoral immune responses as determined inELISA assays during the studies as described in example 3 and 4.

TABLE 9 Overview of ELISA titers in clinical studies Ad26/ Ad26/ Ad26/Ad26/ MVA/ MVA/ MVA/ MVA/ Study MVA MVA MVA MVA Ad26 Ad26 Ad26 Ad26 Day0, 14 0, 28 0, 28 0, 56 0, 14 0, 28 0, 28 0, 56 Study 1001 1001 10021001 1002 1001 1002 1001 d8 22 (13) 18 (0) 20 (7) 22 (0) 19 (7) 18 (0)22 (0) 21 (0) d15 164 (79)* — — — 22 (13)* — — — d22 298 (83) — — — 293(87) — — — d29 — 533 (93)* 477 (100)* 582 (100)* — 36 (40)* 55 (47)* 22(7) d36 915 (100) 946 (100) 965 (100) — 4418 (100) 269 (80) 1025 (93) —d50 — 4274 (100) 2976 (100) — — 10573 (100) 6987 (100) — d57 — — — 854(100)* — — — 21 (7)* d64 — 1554 (100) — — — 568 (100) d78 — 7553 (100) —— — 18729 (100) The data are presented as geometric mean concentration(GMC) in ELISA per mL. Percentage of responders at each time points isindicated in brackets;. Ad26: Immunization with Ad26.ZEBOV; MVA:Immunization with MVA-BN-Filo; Prime boost schedule is indicated inheaders. 0, 14: 14 days interval between prime and boost immunizations;0, 28: 28 days interval between prime and boost immunizations; 0, 56: 56days interval between prime and boost immunizations; *day of boost; GMC:Geometric Mean Concentration. Study 1001 was described in example 3 andstudy 1002 was described in example 4.

Table 10 is a summary of the cellular immune responses as determined inELISpot assays during the studies as described in example 3 and 4.

TABLE 10 Overview of cellular immune responses in clinical studies asdetermined by ELISpot Ad26/ Ad26/ Ad26/ MVA/ MVA/ MVA/ Study MVA MVA MVAAd26 Ad26 Ad26 Day 0, 14 0, 28 0, 56 0, 14 0, 28 0, 56 Study 1001 10011001 1002 1001 1001 d8 25 (13) 25 (7) 25 (7) 25 (13) 25 (0) 25 (0) d15113 (79)* — — 52 (20)* — — d22 354 (75) — — 293 (87) — — d29 — 103 (60)*58 (50) — 25 (7)* 25 (0) d36 203 (92) 463 (87) — 552 (100) 882 (93) —d50 — 390 (79) — — 455 (73) — d57 — — 243 (86)* — — 25 (0)* d64 — — 648(86) — — 440 (100) d78 — — 464 (100) — — 238 (87) Data are representedas median SFU/10⁶ PBMC. Percentage of responders at each time points isindicated in brackets; Ad26: Immunization with Ad26.ZEBOV; MVA:Immunization with MVA-BN-Filo; Prime boost schedule is indicated inheaders. 0, 14: 14 days interval between prime and boost immunizations:0, 28: 28 days interval between prime and boost immunizations; 0, 56: 56days interval between prime and boost immunizations; *day of boost; SFU:Spot Forming Units; PBMC: Peripheral blood mononuclear cells. Study 1001was described in example 3 and study 1002 was described in example 4.

Table 11 is a summary of CD4+ T cell responses as determined byintracellular cytokine staining (ICS) during the studies as described inexample 3 and 4.

TABLE 11 Overview of the CD4+ T cell immune response as measured by ICSin clinical studies Stud- Ad26/ Ad26/ Ad26/ MVA/ MVA/ MVA/ y MVA MVA MVAAd26 Ad26 Ad26 Day 0, 14 0, 28 0, 56 0, 14 0, 28 0, 56 Stud- 1001 10011001 1002 1001 1001 y d8 0.02 (0) 0.02 (0) 0.02 (0) 0.02 (0) 0.02 (0)0.02 (0) d15 0.06 — — 0.02 (7)* — — (36)* d22 0.06 (45) — — 0.15 (73) —— d29 — 0.07 (43)* 0.06 — 0.02 0.02 (7) (31) (13)* d36 0.08 (67) 0.11(64) — 0.19 (73) 0.37 (93) — d50 — 0.15 (60) — — 0.16 (67) — d57 — —0.05 — — 0.02 (0)* (36)* d64 — — 0.16 — — 0.17 (67) (71) d78 — — 0.12 —— 0.08 (53) (57) Data are represented as median total CD4+ cytokineresponse in %. Percentage of responders at each time points is indicatedin brackets; Ad26: Immunization with Ad26.ZEBOV; MVA: Immunization withMVA-BN-Filo; Prime boost schedule is indicated in headers. 0, 14: 14days interval between prime and boost immunizations; 0, 28: 28 daysinterval between prime and boost immunizations; 0, 56: 56 days intervalbetween prime and boost immunizations; *day of boost. Study 1001 wasdescribed in example 3 and study 1002 was described in example 4.

Table 12 is a summary of CD8+ T cell responses as determined byintracellular cytokine staining (ICS) during the studies as described inexample 3 and 4.

TABLE 12 Overview of the CD8+ T cell immune response as measured by ICSin clinical studies Stud- Ad26/ Ad26/ Ad26/ MVA/ MVA/ MVA/ y MVA MVA MVAAd26 Ad26 Ad26 Day 0, 14 0, 28 0, 56 0, 14 0, 28 0, 56 Stud- 1001 10011001 1002 1001 1001 y d8 0.02 (0) 0.02 (0) 0.02 (0) 0.02 0.02 (0) 0.02(0) (0) d15 0.02 — — 0.02 — — (29)* (0)* d22 0.15 (64) — — 0.19 — — (73)d29 — 0.12 0.05 (57) — 0.02 (0)* 0.02 (0) (57)* d36 0.07 (50) 0.27 (67)— 0.34 0.08 (53) — (73) d50 — 0.27 (73) — — 0.01 (53) — d57 — — 0.19 — —0.02 (0)* (86)* d64 — — 0.24 (86) — — 0.07 (53) d78 — — 0.24 (79) — —0.06 (47) Data are presented as median total CD8+ cytokine response in%. Percentage of responders at each time points is indicated inbrackets;. Ad26: Immunization with Ad26.ZEBOV; MVA: Immunization withMVA-BN-Filo; Prime boost schedule is indicated in headers. 0, 14: 14days interval between prime and boost immunizations: 0, 28: 28 daysinterval between prime and boost immunizations; 0, 56: 56 days intervalbetween prime and boost immunizations: *day of boost. Study 1001 wasdescribed in example 3 and study 1002 was described in example 4.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

SEQUENCE LISTING

Glycoprotein Ebola virus Zaire, strain Mayinga (Amino Acid sequence):SEQ ID NO: 1 MGVTGILQLPRDRFKRTSFFLWVIILFQRTFSIPLGVIHNSTLQVSDVDKLVCRDKLSSTNQLRSVGLNLEGNGVATDVPSATKRWGFRSGVPPKVVNYEAGEWAENCYNLEIKKPDGSECLPAAPDGIRGFPRCRYVHKVSGTGPCAGDFAFHKEGAFFLYDRLASTVIYRGTTFAEGVVAFLILPQAKKDFFSSHPLREPVNATEDPSSGYYSTTIRYQATGFGTNETEYLFEVDNLTYVQLESRFTPQFLLQLNETIYTSGKRSNTTGKLIWKVNPEIDTTIGEWAFWETKKNLTRKIRSEELSFTVVSNGAKNISGQSPARTSSDPGTNTTTEDHKIMASENSSAMVQVHSQGREAAVSHLTTLATISTSPQSLTTKPGPDNSTHNTPVYKLDISEATQVEQHHRRTDNDSTASDTPSATTAAGPPKAENTNTSKSTDFLDPATTTSPQNHSETAGNNNTHHQDTGEESASSGKLGLITNTIAGVAGLITGGRRTRREAIVNAQPKCNPNLHYWTTQDEGAAIGLAWIPYFGPAAEGIYIEGLMHNQDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLLQRWGGTCHILGPDCCIEPHDWTKNITDKIDQIIHDFVDKTLPDQGDNDNWWTGWRQWIPAGIGVTGVIIAVIALFCICKFVF Glycoprotein Ebola virus Sudan, strain Gulu(Amino Acid sequence): SEQ ID NO: 2MGGLSLLQLPRDKFRKSSFFVWVIILFQKAFSMPLGVVTNSTLEVTEIDQLVCKDHLASTDQLKSVGLNLEGSGVSTDIPSATKRWGFRSGVPPKVVSYEAGEWAENCYNLEIKKPDGSECLPPPPDGVRGFPRCRYVHKAQGTGPCPGDYAFHKDGAFFLYDRLASTVIYRGVNFAEGVIAFLILAKPKETFLQSPPIREAVNYTENTSSYYATSYLEYEIENFGAQHSTTLFKIDNNTFVRLDRPHTPQFLFQLNDTIHLHQQLSNTTGRLIWTLDANINADIGEWAFWENKKNLSEQLRGEELSFEALSLNETEDDDAASSRITKGRISDRATRKYSDLVPKNSPGMVPLHIPEGETTLPSQNSTEGRRVGVNTQETITETAATIIGTNGNHMQISTIGIRPSSSQIPSSSPTTAPSPEAQTPTTHTSGPSVMATEEPTTPPGSSPGPTTEAPTLTTPENITTAVKTVLPQESTSNGLITSTVTGILGSLGLRKRSRRQTNTKATGKCNPNLHYWTAQEQHNAAGIAWIPYFGPGAEGIYTEGLMHNQNALVCGLRQLANETTQALQLFLRATTELRTYTILNRKAIDFLLRRWGGTCRILGPDCCIEPHDWTKNITDKINQIIHDFIDNPLPNQDNDDNWWTGWRQWIPAGIGITGIIIAIIALLCVCKLLC Glycoprotein Marburg virus Angola (Amino Acidsequence): SEQ ID NO: 3MKTTCLLISLILIQGVKTLPILEIASNIQPQNVDSVCSGTLQKTEDVHLMGFTLSGQKVADSPLEASKRWAFRAGVPPKNVEYTEGEEAKTCYNISVTDPSGKSLLLDPPTNIRDYPKCKTIHHIQGQNPHAQGIALHLWGAFFLYDRIASTTMYRGKVFTEGNIAAMIVNKTVHKMIFSRQGQGYRHMNLTSTNKYWTSSNGTQTNDTGCFGTLQEYNSTKNQTCAPSKKPLPLPTAHPEVKLTSTSTDATKLNTTDPNSDDEDLTTSGSGSGEQEPYTTSDAATKQGLSSTMPPTPSPQPSTPQQGGNNTNHSQGVVTEPGKTNTTAQPSMPPHNTTTISTNNTSKHNLSTPSVPIQNATNYNTQSTAPENEQTSAPSKTTLLPTENPTTAKSTNSTKSPTTTVPNTTNKYSTSPSPTPNSTAQHLVYFRRKRNILWREGDMFPFLDGLINAPIDFDPVPNTKTIFDESSSSGASAEEDQHASPNISLTLSYFPKVNENTAHSGENENDCDAELRIWSVQEDDLAAGLSWIPFFGPGIEGLYTAGLIKNQNNLVCRLRRLANQTAKSLELLLRVTTEERTFSLINRHAIDFLLARWGGTCKVLGPDCCIGIEDLSRNISEQIDQIKKDEQKEGTGWGLGGKWWTSDWGVLTNLGILLLLSIAVLIALSCICRIFTKYIGGlycoprotein Marburg virus Musoke (Amino Acid sequence): SEQ ID NO: 4MKTTCFLISLILIQGTKNLPILEIASNNQPQNVDSVCSGTLQKTEDVHLMGFTLSGQKVADSPLEASKRWAFRTGVPPKNVEYTEGEEAKTCYNISVTDPSGKSLLLDPPTNIRDYPKCKTIHHIQGQNPHAQGIALHLWGAFFLYDRIASTTMYRGKVFTEGNIAAMIVNKTVHKMIFSRQGQGYRHMNLTSTNKYWTSSNGTQTNDTGCFGALQEYNSTKNQTCAPSKIPPPLPTARPEIKLTSTPTDATKLNTTDPSSDDEDLATSGSGSGEREPHTTSDAVTKQGLSSTMPPTPSPQPSTPQQGGNNTNHSQDAVTELDKNNTTAQPSMPPHNTTTISTNNTSKHNFSTLSAPLQNTTNDNTQSTITENEQTSAPSITTLPPTGNPTTAKSTSSKKGPATTAPNTTNEHFTSPPPTPSSTAQHLVYFRRKRSILWREGDMFPFLDGLINAPIDFDPVPNTKTIFDESSSSGASAEEDQHASPNISLTLSYFPNINENTAYSGENENDCDAELRIWSVQEDDLAAGLSWIPFFGPGIEGLYTAVLIKNQNNLVCRLRRLANQTAKSLELLLRVTTEERTFSLINRHAIDFLLTRWGGTCKVLGPDCCIGIEDLSKNISEQIDQIKKDEQKEGTGWGLGGKWWTSDWGVLTNLGILLLLSIAVLIALSCICRIFTKYIG Nucleoprotein Ebola virus TaïForest/Ivory coast (Amino Acid sequence): SEQ ID NO: 5MESRAHKAWMTHTASGFETDYHKILTAGLSVQQGIVRQRVIQVHQVTNLEEICQLIIQAFEAGVDFQESADSFLLMLCLHHAYQGDYKQFLESNAVKYLEGHGFRFEVRKKEGVKRLEELLPAASSGKSIRRTLAAMPEEETTEANAGQFLSFASLFLPKLVVGEKACLEKVQRQIQVHSEQGLIQYPTAWQSVGHMMVIFRLMRTNFLIKFLLIHQGMHMVAGHDANDAVIANSVAQARFSGLLIVKTVLDHILQKTEHGVRLHPLARTAKVKNEVNSFKAALSSLAQHGEYAPFARLLNLSGVNNLEHGLFPQLSAIALGVATAHGSTLAGVNVGEQYQQLREAATEAEKQLQKYAESRELDHLGLDDQEKKILKDFHQKKNEISFQQTTAMVTLRKERLAKLTEAITSTSLLKTGKQYDDDNDIPFPGPINDNENSEQQDDDPTDSQDTTIPDIIVDPDDGRYNNYGDYPSETANAPEDLVLFDLEDGDEDDHRPSSSSENNNKHSLTGTDSNKTSNWNRNPTNMPKKDSTQNNDNPAQRAQEYARDNIQDTPTPHRALTPISEETGSNGHNEDDIDSIPPLESDEENNTETTITTTKNTTAPPAPVYRSNSEKEPLPQEKSQKQPNQVSGSENTDNKPHSEQSVEEMYRHILQTQGPFDAILYYYMMTEEPIVFSTSDGKEYVYPDSLEGEHPPWLSEKEALNEDNRFITMDDQQFYWPVMNHRNKFMAILQHHK

1. A method of enhancing an immune response in a human subject, themethod comprising: a. administering to the human subject a firstcomposition comprising an immunologically effective amount of an MVAvector comprising a first polynucleotide encoding an antigenic proteinor an immunogenic polypeptide thereof for priming the immune response;and b. administering to the subject a second composition comprising animmunologically effective amount of an adenovirus vector comprising asecond polynucleotide encoding the antigenic protein or an immunogenicpolypeptide thereof for boosting the immune response; to thereby obtainan enhanced immune response against the antigenic protein in the humansubject.
 2. The method according to claim 1, wherein the enhanced immuneresponse comprises an enhanced antibody response against the antigenicprotein in the human subject.
 3. The method according to claim 1,wherein the enhanced immune response comprises an enhanced CD8+ T cellresponse against the antigenic protein in the human subject.
 4. Themethod according to claim 1, wherein the enhanced immune responsecomprises an enhanced CD4+ T cell response against the antigenic proteinin the human subject.
 5. The method according to claim 1, wherein theenhanced immune response comprises an enhanced CD8+ T cell response oran enhanced CD4+ T cell response, and wherein the enhanced CD8+ or CD4+T cell response comprises an increase or induction of a dominant CD8+ orCD4+ T cell response against the antigenic protein in the human subject.6. The method according to claim 1, wherein the enhanced immune responsecomprises an enhanced CD8+ T cell response or an enhanced CD4+ T cellresponse, and wherein the enhanced CD8+ or CD4+ T cell responsecomprises an increase or induction of polyfunctional CD8+ or CD4+ Tcells specific to the antigenic protein in the human subject.
 7. Themethod according to claim 2, wherein the enhanced immune responsefurther comprises an enhanced CD8+ T cell response and an enhanced CD4+T cell response against the antigenic protein in the human subject. 8.(canceled)
 9. The method according to claim 7, wherein the enhanced CD8+and CD4+ T cell response comprises an increase or induction ofpolyfunctional CD4+ and CD8+ T cells specific to the antigenic proteinin the human subject.
 10. The method according to claim 1, wherein theenhanced immune response comprises an enhanced CD4+ T cell response, anenhanced antibody response and an enhanced CD8+ T cell response, againstthe antigenic protein in the human subject.
 11. The method according toclaim 1, wherein the enhanced immune response provides a protectiveimmunity to the human subject against a disease related to the antigenicprotein.
 12. The method according to claim 1, wherein the adenovirusvector is an rAd26 vector.
 13. The method according to claim 1, whereinstep (b) is conducted 1-12 weeks after step (a).
 14. The methodaccording to claim 1, wherein step (b) is conducted 2-12 weeks afterstep (a).
 15. The method according to claim 1, wherein step (b) isconducted at least 1 weeks after step (a).
 16. The method according toclaim 1, wherein step (b) is conducted at least 2 weeks after step (a).17. The method according to claim 1, wherein the antigenic protein isderived from a pathogen or a tumor.
 18. The method according to claim17, wherein the antigenic protein is derived from a filovirus.
 19. Themethod according to claim 18, wherein the antigenic protein comprisesthe amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO:
 5. 20. Themethod according to claim 19, wherein the MVA vector comprises apolynucleotide encoding the antigenic proteins having the amino acidsequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5.21. The method according to claim 19, wherein the adenovirus vectorcomprises a polynucleotide encoding at least one antigenic proteinhaving the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ IDNO:
 3. 22. The method according to claim 19, wherein the adenovirusvector comprises a polynucleotide encoding the antigenic protein havingthe amino acid sequence of SEQ ID NO:
 1. 23. The method according toclaim 22, wherein the adenovirus vector is an rAd26 vector.
 24. A methodof enhancing an immune response against at least one filovirus subtypein a human subject, comprising: a. administering to the human subject afirst composition comprising an immunologically effective amount of aMVA vector comprising a polynucleotide encoding an antigenic protein ofthe at least one filovirus subtype, a substantially similar antigenicprotein, or an immunogenic polypeptide thereof, for priming the immuneresponse; and b. administering to the subject a second compositioncomprising an immunologically effective amount of ant adenovirus vectorcomprising a polynucleotide encoding an antigenic protein of the atleast one filovirus subtype, a substantially similar antigenic protein,or an immunogenic polypeptide thereof, for boosting the immune response;to thereby obtain an enhanced immune response against the at least onefilovirus subtype in the human subject.
 25. A vaccine combinationcomprising: (a) a first composition comprising an immunologicallyeffective amount of a MVA vector comprising a first polynucleotideencoding an antigenic protein or an immunogenic polypeptide thereof; and(b) a second composition comprising an immunologically effective amountof an adenovirus vector comprising a second polynucleotide encoding theantigenic protein or an immunogenic polypeptide thereof wherein thefirst composition is administered to the human subject for priming theimmune response, and the second composition is administered to the humansubject one or more times for boosting the immune response. 26.(canceled)
 27. The vaccine combination of claim 25, wherein theantigenic protein is derived from a pathogen or a tumor.
 28. The vaccinecombination of claim 25, wherein the antigenic protein is derived from afilovirus.
 29. The vaccine combination of claim 28, wherein theantigenic protein comprises the amino acid sequence selected from thegroup consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO:4, and SEQ ID NO:
 5. 30. The vaccine combination of claim 29, whereinthe MVA vector comprises a polynucleotide encoding the antigenicproteins having the amino acid sequences of SEQ ID NO: 1, SEQ ID NO: 2,SEQ ID NO: 4, and SEQ ID NO:
 5. 31. The vaccine combination of claim 29,wherein the adenovirus vector comprises a polynucleotide encoding atleast one antigenic protein having the amino acid sequence of SEQ ID NO:1, SEQ ID NO: 2, or SEQ ID NO:
 3. 32. The vaccine combination of claim31, wherein the adenovirus vector comprises a polynucleotide encodingthe antigenic protein having the amino acid sequence of SEQ ID NO: 1.33. The vaccine combination of claim 32, wherein the adenovirus vectoris an rAd26 vector.